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Serous cysts of the aging guinea pig ovary. II. Scanning and transmission electron microscopy

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Serous Cysts of the Aging Guinea Pig Ovary
II. SCANNING AND TRANSMISSION
ELECTRON MICROSCOPY
STEVEN L QUATTROPANI
Department ofAnatomy, Medical College of Virginia, Virginia Commonwealth
Uniuersity, Richmond, Virginia 23298
ABSTRACT
Ovarian cysts which developed spontaneously from the rete ovarii in the ovaries of guinea pigs have been examined in l- and 2-year-old animals
by scanning and transmission electron microscopy. Two cell types have been described, a tufted cell with numerous motile cilia and a non-tufted cell with a solitary cilium. While the non-tufted cell exhibited numerous smooth coated vesicles
in its lateral and basal regions, no signs of secretion to the lumen of the cyst have
been seen and occluding junctions have not been observed.
The rete ovarii of guinea pigs older than one
year of age is generally cystic (Young e t al.,
'38; Quattropani, '77). I n a previous report
(Quattropani,'771, i t was stated t h a t each animal examined which was over one year old had
at least one cyst between 1 mm and 2.5 cm in
diameter. The frequency with which these
cysts appeared, t h e correlation of this frequency with age, their origin and their light
microscopic appearance suggested t h a t they
might serve a s a model for the study of t h e
serous cystadenoma of the human. The prese n t work describes t h e cysts of animals one
and two years old using transmission and
scanning electron microscopy. I t serves to extend t h e observations of cell structure made in
t h e earlier report and enables a comparison of
this cyst with t h e serous cystadenoma of
humans.
MATERIALS AND METHODS
The animals used in this study were Hartley
strain guinea pigs. Ovaries from eight l-yearold and five 2-year-old animals were examined
by light and electron microscopy. The animals
were killed either by decapitation or by
sodium pentobarbital injection. The ovaries
were removed and placed in a fixative containing 3% glutaraldehyde and 1%paraformaldehyde and buffered with 0.1 M phosphate buffer
(pH 7.4). Portions of cysts greater than 1 mm
in diameter were removed and processed for
either transmission or scanning electron microscopy. Tissues to be used for transmission
electron microscopy were fixed for two hours
and subsequently immersed in 1%osmium
ANAT. REC. (1978) 190: 285-298.
tetroxide buffered with 0.1 M phosphate buffer
(pH 7.4), dehydrated with alcohol, and embedded in Epon 812. Semi-thin sections (0.75 p m )
were stained with toluidine blue for light microscopic examination. Thin sections for electron microscopy were stained with uranyl acet a t e and lead citrate. For scanning electron
microscopy tissues from two animals in each
age group were dehydrated in acetone after
aldehyde fixation and subsequently critical
point dried with Freon 116. Portions of a cyst
3 mm in diameter were excised prior to exposure to fixatives, placed in modified Hank's
solution (Flow) and examined by phase microscopy.
RESULTS
Since no differences have been detected between the large cysts of the two age groups,
the following descriptions pertain to both
ages. The luminal surface of these cysts examined by scanning microscopy was devoid of epithelial projections or papillae. Tufts of cilia
were present (fig. 1). These tufts were randomly distributed; they might be isolated (fig.
2), or clumped together in any given region.
The cilia of a tuft appeared to be bent to varying degrees; however, generally they were
bent in the same direction. The tufts of a given
region also generally were bent in the same direction. I n addition to the tufts of cilia, two
other cellular projections were seen on this
surface: solitary cilia and microvilli (figs. 2,
3). The solitary cilia were longer than t h e cilia
Received June 8, 'I1 Accepted Aug. 29, '77.
285
286
STEVEN L. QUATTROPANI
found in tufts; they were evenly distributed
and usually arose from a pit or depression.
This indentation of the cell surface was generally in a region which was devoid of other
surface projections and was bordered by a
ridge of microvilli. The microvilli also were
present in t h e area surrounding the tuft of
cilia and interspersed among these cilia. No
signs of secretory activity were detected.
Two cell types have been identified by light
microscopy: the tufted cell and the non-tufted
cell (Quattropani, '77). The non-tufted cell
was a low columnar to squamous cell with a
single cilium projecting into the lumen. The
nucleus, which by light microscopy appears
spherical in semi-thin sections, by electron
microscopy was seen to be irregularly shaped,
often with deep clefts. Its nucleolus was composed of a pars amorpha and nucleonema. Heterochromatin lined most of t h e peripheral region of the nucleus, and a few nuclear pores
were present in its nuclear envelop (fig. 4).
The basal portion of the cell (fig. 5 ) rested on a
basal lamina. The cytoplasm immediately
adjacent to the plasmalemma in t h a t region
exhibited patches of greater electron opacity
(fig. 5) due to t h e presence of a layer of fibrillar material (fig. 5 inset). Smooth surfaced
vesicles were present adjacent to this membrane and in varying degrees of continuity
with it. Coated vesicles were also present but
less abundant. The lateral borders between
non-tufted cells exhibited interdigitation, invaginations of t h e plasmalemma and smooth
surfaced vesicles (fig. 4).In the juxta-luminal
region of this epithelium, a junctional complex linked adjacent cells. This complex was
composed of a zonula adherens and a macula
adherens (figs. 6-81. Gap junctions (fig. 8 )
were also occasionally observed, but not occluding junctions. The luminal surface of t h e
cell had microvilli and a solitary cilium. The
microvilli were approximately 1 p m long and
were most numerous a t the periphery of this
luminal surface (fig. 6 ) .The ciliary shaft had
concentrically arranged tubular elements. A
central doublet was identified in cross sections of cilia which lay in the depressions of
t h e non-tufted cells (fig. 10). Occasionally
cross sections of cilia within t h e lumen were
seen which exhibited nine tubular elements
irregularly spaced (fig. 9). These elements
were both singlets and doublets. Such profiles
could be seen in regions devoid of tufted cells.
Within the cell, the cilium terminated in a
basal body from which a striated rootlet pro-
jected, Adjacent to this basal body was a centriole. The cytoplasm of this cell contained
elongate mitochondria with lamellar cristae
(figs. 4, 6 ) . The endoplasmic reticulum was
tubular and sparsely studded with ribosomes
(figs. 5 , 6 ) . Free and polyribosomes as well as
microtubules and microfilaments were found
randomly distributed throughout the cell. Microfilaments and fibrillar material at the apical surface formed a band t h a t ended in the region of the zonula adherens (figs. 6-81, Elements of the Golgi complex were found in the
supranuclear and lateral regions of the cell
and were composed of four to six flattened
cisternae and smooth and coated vesicles (fig.
4). In t h e basal portion of t h e cell, membrane
bound granules with a homogeneous electron
opaque core were seen (fig. 5 inset).
The tufted cell was distinguished by the
concentration of cilia on its luminal surface
(fig. 11). These cells were generally bordered
laterally by non-tufted cells to which they
were bound by zonulae adherentes. The tufted
cell was a columnar cell the luminal surface of
which might be flat or domed. In its basal region was a large nucleus similar in appearance
to t h a t of t h e non-tufted cell. The lateral
borders of the tufted cell, while generally
straight, did exhibit some interdigitation with
neighboring non-tufted cells. A small portion
of the basal surface of t h e cell, generally less
than one-third of t h e cell's width, rested on
the basal lamina (fig. 13). Foot processes of
adjacent non-tufted cells intervened in the
rest of this region. The cytoplasm of the tufted
cell (fig. 11) contained mitochondria, granular
and agranular endoplasmic reticulum similar
to those of the non-tufted cell. The Golgi complex, while similar in appearance to that of
t h e non-tufted cell, was generally supranuclear. Mitochondria also were more numerous
in the supranuclear region and one or more
multivesicular bodies also were seen in this
area.
Microvilli as well a s cilia projected from
t h e luminal surface. Although the microvilli
might be found between cilia, they were most
common a t the periphery of the luminal surface of t h e cell. The shaft of the cilia had a
complement of nine peripheral doublets and
two central tubular elements; at its distal end
t h e shaft decreased in diameter and exhibited
11 equally spaced tubular elements (fig. 12).
In t h e more basal portion of the shaft (fig. 11)
the region within t h e circle of doublets had a
greater electron opacity. This area extended
SEROUS CYSTS OF THE AGING GUINEA PIG OVARY
from approximately 0.4-0.2p m from t h e point
where the shaft joined the plasmalemma between cilia. The central tubular elements terminated in the proximal end of this region.
The plasmalemma of the transitional region
at the base of t h e cilium had focal thickenings
(fig. 11). Within the cell t h e cilium terminated a basal body from which projected laterally a foot process and a striated rootlet.
Light microscopic examination of Epon sections demonstrated t h e presence of cells which
were refractile to staining with toluidine blue.
These clear cells were few in number and their
distribution irregular. Although most specimens exhibited them, they were not found in
all samples of a given specimen. Serial sections of some of t h e clear cells indicated tufts
of cilia similar to those of the tufted cell. Electron microscopy confirmed the presence of the
cilia (fig. 14) in some of these cells although
some clear cells were devoid of cilia. The nuclear envelope and endoplasmic reticulum of
these clear cells were dilated and there was a
reduction in t h e concentration of mitochondria and elements of endoplasmic reticulum,
the Golgi complex was not a s well developed
and the cytoplasmic matrix was more electron
lucent than t h a t of t h e tufted or non-tufted
cells. Lateral interdigitations of these cells
were reduced and pinocytotic vesicles were
absent.
A portion of t h e wall of a 3-mm cyst from a
1-year-old animal was placed in modified
Hank's solution prior to fixation and examined by phase microscopy. The cilia of t h e
tufted cells were beating actively and in a
given field exhibited a metachronal pattern.
The solitary cilia could not be seen in these
preparations.
DISCUSSION
In this tissue two primary cell types have
been described, the tufted cell and the nontufted cell. They a r e distinguished from one
another by apical and basal specializations,
the number and distribution of cilia and microvilli, and t h e presence of cellular foot processes. A third cell type, t h e clear cell, is considered a variant of one or the other of these
cell types depending on t h e presence or absence of cilia. While such clear cells might be
considered as being due to poor fixation, t h e
presence of well fixed cells adjacent to them
suggests t h a t their appearance is not a n
artifact. Cells with a similar light microscopic
appearance have been described in t h e oviduct
287
and identified as deciliated or mitotic cells
(Brenner, '69; Odor and Blandau, '73). However, no signs of ciliogenesis or mitosis have
been seen in these cysts. The morphology of
t h e clear cells in the guinea pig cyst suggests
that they do not function as the typical tufted
or non-tufted cells and may in fact be degenerative cells. I t should be noted that while the
tissues for this study were taken only during
diestrus, Young e t al. ('38) in their study of
the guinea pig ovarian cycle stated t h a t they
found no evidence of rhythmic regression of
rete cysts. We conclude t h a t these clear cells
represent a small population which are in t h e
process of cell death.
The cilia of t h e tufted cell are similar in
structure to cilia which have been observed
in other epithelia of t h e reproductive tract
(Ferenczy and Richart, '74). They have the
nine peripheral doublets and central pair of
singlets considered characteristic of motile
cilia (Satir, '68). They also have a ciliary
necklace in t h e transitional portion of the
shaft (Gilula and Satir, '72). In the distal region of the shaft, there is the transition of the
doublets to singlets which has been described
by Satir ('68). Cross-sections of the tips of
these cilia are distinguished from those of the
non-tufted cell by the presence of 11 singlets.
Direct observation confirms t h a t these cilia
a r e motile and t h a t their movement is coordinated. Since occluding junctions are not present, the zonula adherens, which is in the region of t h e apical band of microfilaments,
macula adherens and the occasional gap junction are t h e only structural components which
might be involved in this coordination.
The solitary cilium of the non-tufted cell
like those found in a variety of other tissues
(McCarron and Anderson, '73; Rash e t al., '69)
has a configuration different from the motile
cilia of t h e tufted cells. When observed, the
central element is a doublet rather than two
singlets, and may be one of the nine peripheral
doublets which has been displaced centrally.
In none of the sections have 11 elements been
observed. Such central displacement has been
described in neurons (Allen, '65). Cilia which
exhibit a departure from the 9 + 2 pattern
have been considered non-motile (Fawcett,
'61). However, motile forms with the 9 + O configuration have been noted (Afzelius, '62;
Stubbelfield and Brinkley, '661, and according
to Blandau ('77) the solitary cilia of the
oviduct are also motile.
Analysis of t h e ultrastructure of this tissue
288
STEVEN L. QUATTROPANI
makes apparent several questions concerning
the function of this cyst. The presence of
motile cilia in this closed system could be explained by the fact t h a t the rete ovarii and
oviduct have a common embryonic origin.
Questions pertaining to the mechanism by
which the cyst is maintained and the fluid
within the cyst is elaborated are not a s easily
answered. Several factors could facilitate the
maintenance of t h e vesicular form of the
cyst: intercellular and intracellular specializations, a rigid lamina propria and fluid
pressure. Intercellularjunctions, microtubules
and microfilaments have been observed in the
cyst cells; however, neither they nor the lamina propria (Quattropani, ’771, appear to be
sufficiently developed to account for cyst
structure. The collapse of large cysts after experimental rupture suggests that hydrostatic
pressure may serve this function; however,
the mechanism of fluid retention is not
known. The basal lamina of the cyst epithelium is not thick and while the epithelial cells
interdigitate, no occluding junctions have
been seen. In spite of the presence of a well developed Golgi complex, no secretory granules
have been found in the cyst cells and the
mechanism of fluid elaboration is unknown.
The cells do, however, appear to be adapted for
uptake of extracellular material. Lateral interdigitation and foot processes provide increased surface area which may facilitate absorption. I t is of interest t h a t the non-tufted
cell by virtue of its lateral interdigitation and
foot processes has a greater surface area than
the tufted cell which has few of these processes and is partially shielded from the basal
surface by those of the non-tufted cell. This
difference in surface may be indicative of
functional specialization of t h e two cell types.
The lateral and basal regions of both types
have numerous smooth coated caveolae and
vesicles, suggesting a n active uptake of extracellular material. The fate of these vesicles
presently is unknown. Similar vesicles are
seen in the Golgi complex but few vesicular
elements are seen in the apical region of the
cell.
The appearance of the cytoplasm of the
guinea pig cyst cells is in general agreement
with the description of the benign serous
cystadenoma of t h e human ovary as presented
by Gondos (’71). The mitochondria, endoplasmic reticulum and Golgi complex of these two
tissues are similar in appearance. Both tissues
exhibit cilia with the 9 + 2 configuration, microvilli, terminal bars, and interdigitation of
t h e plasma membrane. However, t h e absence
of secretory granules in the guinea pig cyst
and the apparent absence of solitary cilia and
micropinocytotic vesicles may represent significant differences between the two tissues.
The similarities in the gross appearance and
light microscopy of this cyst with that of human ovarian cystomas prompted the present
ultrastructural study. We have seen that at
this level of investigation there were additional morphological similarities. As a result
of this study, however, several questions concerning the function of the cyst epithelium
arose. These questions are currently under
investigation.
LITERATURE CITED
Afzelius, B. A. 1962 The contractile apparatus in some
invertebrate muscles and spermatozoa. In: Proceedings of
the Fifth International Congress for Electron Microscopy. S. S. Breese, J r . , ed. Academic Press, New York, Vol. 2,
p. M-1.
Allen, R. A. 1965 Isolated cilia in inner retinal neurons
and in retinal pigment epithelium. J. Ultrastruct. Res.,
12: 730-747.
Blandau, R. J. 1977 (personal communication)
Brenner, R. M. 1969 Renewal of oviduct cilia during the
menstrual cycle of the Rhesus monkey. Fertil. Steril.,20:
599-611.
Fawcett, D. 1961 Cilia and flagella. In: The Cell. J.
Brachet and A. E. Mirsky, eds. Academic Press, New
York, Vol. 11, pp. 217-297.
Ferenczy, A., and R. M. Richart 1974 Female Reproductive
System. Dynamics of Scan and Transmission Electron
Microscopy. John Wiley and Son, New York.
Gilula, N. B., and P. Satir 1972 The ciliary necklace. A ciliary membrane specialization. J. Cell Biol., 53: 494-509.
Gondos, B. 1971 Electron microscopic study of papillary
serous tumors of the ovary. Cancer, 27: 1455-1464.
McCarron, L. K., and E. Anderson 1973 A cytological study
of the postnatal development of the rabbit oviduct epithelium. Biol. Reprod., 8: 11-28.
Odor, D. L., and R. J.Blandau 1973 Egg transport over the
fimbrial surface of the rabbit oviduct under experimental
conditions. Fertil. Steril., 24: 292-300.
Quattropani, S. L. 1977 Serous cysts of the aging guinea
pig. I. Light microscopy and origin. Anat. Rec., 288:
351-360.
Rash, J. E., J. W. Shay and J.J.Biesele 1969 Cilia in cardiac differentiation. J. Ultrastruct. Res., 29: 470-484.
Satir, P. 1968 Studies on cilia. 111. Further studies on
the cilium tip and a “sliding filament” model of ciliary
motility. J. Cell Biol., 39: 77-94.
Stubbelfield, E., and B. R. Brinkley 1966 Cilia formation in
Chinese hamster fibroblast in uitro as a response to colcemid treatment. J. Cell Biol., 30; 645-652.
Young, W. C., E. W. Dempsey, H. I. Myers and C. W. Hagquist 1938 The ovarian condition and sexual behavior of
the female guinea pig. Am. J. Anat., 63: 457-483.
PLATES
PLATE I
EXPLANATION OF FIGURES
1 A scanning electron micrograph of the lurninal surface of a cyst. Several tufts of cilia
as well as solitary cilia are seen. X 2,600.
2
A tuft of cilia surrounded by rnicrovilli.
X
5,000
3 The base of one of these solitary cilia lies in a pit (>1. Microvilli are not numerous in
the region immediately surrounding the base of the cilium but are clustered in a ridgelike configuration. X 5,000.
SEROUS CYSTS OF THE AGING GUINEA PIG OVARY
Steven L. Quattropani
PLATE 1
29 1
PLATE 2
EXPLANATION OF FIGURES
4 Portions of two non-tufted cells are seen in this micrograph. Note the well developed
Golgi complex IG), the lateral interdigitation of these cells, the presence of smooth
surfaced caveolae vesicles I > ) and the nuclear pore (P). X 35,000.
5 Basal region of a non-tufted cell with a basal lamina (BL), smooth surfaced caveolae
(> ),and sparsely granulated endoplasmic reticulum IER). The inset is of another nontufted cell and shows the membrane bound granules (D)and fihrillar material (F)
adjacent to the plasmalemma. X 44,500. Inset X 35,000.
292
SEROUS CYSTS OF THE AtiING GUINEA PIG OVARY
Steven L. Quattropani
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
6 Apical portions of two non-tufted cells showing microvilli (M), mitochondria with lamellar cristae, sparsely granulated endoplasmic reticulum (ER) and a zonula adherens
( > ) . X 46,000.
7
A rnacula adherens
8 A gap junction
294
( >)
between two non-tufted cells.
( 2 )between
two non-tufted cells.
X
X
36,000.
36,000.
SEROUS CYSTS OF T H E AGING GUINEA PIG OVARY
Steven L. Quattropani
PLATE 3
295
PLATE 4
EXPLANATION OF FIGURES
9 A section through the shaft of a cilium fron a non-tufted cell showing nine tubular
elements. X 32,000.
10 A portion of a cilium is seen in the pit of this non-tufted cell. Note the central doublet
and the presence of nine tubular elements. X 35,000.
11 This apical portion of a tufted cell shows several cilia with basal bodies. Note the ciliary necklace (> 1 and the area of increased electron opacity between bars. X 24,000.
12 Cilia of a tufted cell are seen here cut a t various positions along the shaft. Note the
presence of the central pair of singlets and the total of 11tubular elements in a given
cross section. X 35,000.
296
SEROUS CYSTS OF THE AGING GUINEA PIG OVARY
Steven L Quattropani
PLATE 4
297
SEROUS CYSTS OF THE AGING GUINEA PIG OVARY
Steven L. Quattropani
EXPLANATION OF FIGURES
13 The basal portion of a tufted cell bordered by two non-tufted cells. The area between
the bars is that portion of the cell which lies on the basal lamina. x 19,000,
14 A tufted clear cell. Note the paucity of organelles and the electron lucent cytoplasmic
matrix. x 9,000.
PLATE 5
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