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Intracellular collagen in the nonpregnant and IUD-containing rat uterus.

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lntracellular Collagen in the Nonpregnant and
IUD-containing Rat Uterus
Departments of Anatomy and Obstetrics and Gynccology, Louisiana State
Unzversity Medical Center, New Orleans, Louzstana 70112
An experiment designed to study the effects of the copper IUD
on the virgin rat uterus has revealed the presence of intracellular collagen fibrils
in control uteri and in uteri that have contained a copper IUD for three months.
The cells containing the collagen are found in the stroma i n close proximity to
the uterine epithelium. The collagen is found within membrane-bound cytoplasmic vacuoles that vary in morphology. In some cases the fibrils are tightly packed
and linear, with no other material evident i n the vacuole. In other examples,
the fibrils are randomly arranged and the vacuoles contain a punctate material
which is characteristic of phagolysosomes. Finally, cytoplasmic vacuoles are
seen which contain ill-defined debris and poorly-visualized structures that exhibit a periodicity, suggesting a terminal phase of fibril breakdown. All animals
were sacrificed in metestrus, and the results indicate that intracellular collagen
is present in the nulliparous rat uterus at this stage of the cycle. In addition,
this phenomenon does not appear to be influenced by the presence of a copper
IUD over a period of three months
In recent years electron microscope
studies have documented the presence of
collagen fibrils within the cytoplasm of a
variety of connective tissue cells. For example, intracellular collagen has been
demonstrated in granuloma tissue (PerezTamayo, '70); in fibroblasts of scar tissue
(Ten Cate and Freeman, '74; Dyer and
Enna, '75), periodontal ligament (Ten Cate,
'72; Deporter and Ten Cate, '73; Listgarten, '73; Ten Cate and Deporter, '74; Eley
and Harrison, '75), and tissue overlying
non-erupted teeth (Ten Cate and Syrbu,
'74); in tumor cells of chondrosarcoma
(Imura et al., '75); and in cells of the postpartum involuting uterus (Luse and Hutton, '64; Schwarz and Gulden, '67; Brandes
and Anton, '69; Parakkal, '69). The presence of intracellular collagen is most evident in those systems where collagen remodelling is occurring, and is considered
to be a mechanism for the degradation of
collagen material (Parakkd, '69; Ten Cate
and Freeman, '74).
The postpartum involuting uterus has
provided a good model for studying collagen
breakdown and for demonstrating intracellular collagen fibrils. To our knowledge
only one electron microscope study included
examination of control non-pregnant uteri,
REC.,187. 241-248.
and in this instance no mention was made
of intracellular collagen fibrils (Parakkal,
'69). We have been studying the effects of
copper I U D s on the ultrastructure of the
nulliparous rat uterus. The connective tissue underlying the uterine epithelium of
control and experimental animals was examined in order to determine if intracellular collagen is present in the non-pregnant
uterus, and if so, the effects of the implanted IUD on this phenomonon. This paper reports the results of the investigation.
Nulliparous female rats (Holtzman) were
obtained at 60 to 66 days of age and maintained under conditions of controlled lighting (fluorescent illumination from 5 A . M .
to 7 P.M.) and temperature (22 +- 2'c).
Free access was provided to laboratory chow
and tap water. Daily vaginal smears were
taken and after three consecutive estrous
cycles, a segment of pure copper wire was
inserted into alternate left or right uterine
horns at metestrus (vaginal smear of leuReceived June 3, '76. Accepted Sept. 9, '76.
1 Supported by Edward G. Schlieder Foundation Grant.
The authors thank Jane11 Buck and JoAnn Canale
for their technical assistance, Mary Ann Anderson for
her secretarial services, and Garbis Kerimian for h i s
excellent photographic work.
24 1
cocytes and nucleated epithelial cells which
followed a smear of all cornified cells).
The insertion of the wire was done in accordance to the technique of Chang and
Tatum ('72), except for one modification,
which was that each end of the wire was
coiled separately rather than being twisted
Experimental animals were killed at
metestrus three months (155-163 days of
age) after the insertion of the wire segment. Control rats were killed at this time
as well. It should be noted that the regularity of the estrous cycle and the vaginal
smear pattern began to change in rats
with the wire segment after two months.
The length of the cycle became longer, and
the daily smears showed an increase in
leucocytes and a decrease in cornified cells
with respect to frequency and number
(Peppler , '75).
Segments of uterus in the form of short
cylinders, from both control and experimental animals, were placed in cold 4%
glutaraldehyde buffered to pH 7.2 with
0.2 M phosphate buffer, fixed for at least
24 hours, and rinsed in 0.2 M phosphate
buffer containing 0 . 3 M sucrose. The tissues were post-fixed for two hours in 1%
osmium tetroxide buffered to pH 7.2 with
0.2 M phosphate buffer, dehydrated in a
graded series of alcohol, and equilibrated
with propylene oxide. The tissues were embedded flat in Spurr embedding medium,
in order to facilitate orientation and crosssectional sampling of the material. Thin
sections were cut on a Reichert OMU2 ultramicrotome, stained with lead and uranyl
ions, and viewed with a Philips EM 200
electron microscope.
Examination of both control uteri and
uteri containing a copper IUD for a three
month period revealed small numbers of
cells that contained collagen fibrils within
the cytoplasm. The frequency of appearance and the morphology of' intracellular
collagen were similar in both cases, and
the description that follows is applicable to
both control and experimental systems.
In general, intracellular collagen was
found more easily in connective tissue cells
that were in close proximity to the overlying uterine epithelium. In many cases,
these cells were adjacent to the basal la-
mina of the epithelium (fig. 1). whereas
other cells containing intracellular collagen were located in the adjacent deeper
regions of the endometrial stroma.
The cells containing the intracellular
collagen were dissimilar with respect to
ultrastructural features. Some cells contained few mitochondria, small amounts
of rough endoplasmic reticulum, a well
developed Golgi zone, and bodies with the
morphological characteristics of lysosomes
(fig, 1). Other cells were characterized by
numerous mitochondria and abundant
rough endoplasmic reticulum (fig. 2). None
of these various cell types were restricted
to any particular region of the endometrial
The morphology associated with the cytoplasmic collagen was variable. In some
instances banded fibrils were located within
structures considered to be phagosomes.
Limiting membranes were in apposition to
the fibrils that were, in general, linear or
slightly curved (fig. 2). In other examples,
collagen fibrils were obseilred within larger
vacuolar structures that also contained
punctate densities (fig. 3). Segments of the
fibrils within these larger vacuoles were
often convoluted (fig. 3), and occasionally
fibrils were seen arranged circularly at the
periphery of the vacuole (fig. 4). Other
vacuoles contained banded structures that
were barely discernable as collagen fibrils
(fig. 5). Some cells contained multiple aggregates of intracellular collagen, with
each aggregate being different from a morphological standpoint (fig. 6).
Intracellular collagen has been described
in a variety of tissues, in most instances
where extensive collagen remodelling is
occurring. It is generally considered that
this represents one mechanism that is important in the degradation of extracellular
collagen (Parakkal, '69; Ten Cate and
Freeman, '74). Intracellular collagen has
not been previously demonstrated in the
nonpregnant rat uterus. There is relatively
little collagen turnover occurring in this
system, and therefore the opportunity of
viewing intracellular collagen at the electron microscope level is much reduced. It
is known that some collagen turnover occurs in the nonpregnant rat uterus, and is
one tissue where this continues into adult-
hood (Tang Kao et al., '61). Our study supports this concept in that intracellular
collagen has been demonstrated in connective tissue cells of the endometrial stroma
of the adult nulliparous rat uterus. The
morphology of the intracellular collagen is
similar to that described in other studies
and it is assumed that this represents collagenous degradation as described by Ten
Cate and Syrbu ('74). The presence of a
copper IUD for three months does not seem
to enhance or lessen the frequency of this
finding. This suggests that the copper IUD
does not exert any obvious effects on those
cells in the endometrial stroma that are
involved in this process.
The majority of cells containing intracellular collagen are in the region immediately beneath the uterine epithelium. Recent immunohistochemical studies have
demonstrated collagenase activity in the
nonpregnant rat in the basement membrane region of the uterine epithelium
(Montfort and Perez-Tamayo, '75). This
suggests that a basal rate of collagen turnover may be occurring at this site. It would
therefore be expected that cells involved in
the degradation process would be localized
in this area, and this seems to be the case.
The cell types involved in sequestering
and degrading collagen fibrils in the nonpregnant rat uterus cannot be clearly classified as a single population of cells. Other
studies have specified the involved cells as
being fibroblasts or macrophages. The present study has demonstrated cells containing cytoplasmic collagen that could be
classified as macrophages or fibroblasts
based strictly on cell ultrastructure. Such
classification is mainly based on the presence or absence of lysosomes, and plane of
section must be taken into account under
these circumstances. It may be that the
fibroblast, generally considered to be poorly
phagocytic, is capable under certain circumstances of becoming highly phagocytic
and involved in degradative activity. A
similar suggestion has been made with respect to cells that are involved in the healing process of tendon (Greenlee and Pike,
'71). There also exists in the literature a
number of studies demonstrating that one
cell type may be capable of synthesizing
collagen and also containing the necessary
proteins for cell contraction, this cell being
the myofibroblast (Gabbiani et al., '71). The
situation in the rat uterus may be another
example of cell modulation, based perhaps
on hormonal parameters. The uterus is
responsive to specific hormone action, and
since i t is now known that the nonpregnant and involuting uterus differ only with
respect to degree of collagen turnover, it
may be that this system can provide more
insight into the possible hormonal control
of this cellular activity. Studies supporting
this concept have been reported recently
by Tansey and Padykula ('76).
Brandes, D., and E. Anton 1969 Lysosomes in
uterine involution: Intracytoplasmic degradation of myofilaments and collagen. J. Gerontol.,
24: 55-69.
Chang, C. C., and H. J. Tatum 1972 Some temporal relationships between intrauterine copper
wire and its contraceptive action in the rat. Fertil. Steril., 23: 191-195.
Deporter, D. A., and A. R. Ten Cate 1973 Fine
structural localization of acid and alkaline phosphatase in collagen-containing vesicles of fibroblasts. J. Anat., 114: 4 5 7 4 6 1 .
Dyer, R. F., and C. D. Enna 1975 Ultrastructure
of keloid: An unusual incident involving lepromatous leprosy. Int. J. Dermatol., 24: 743-754.
Eley, B. M., and J. D. Harrison 1975 Intracellular collagen fibrils in the periodontal ligament
of man. J. perio. Res., 10: 168-170.
Gabbiani, G., G . B. Ryan and G. Majno 1971
Presence of modified fibroblasts in grandation
tissue and their possible role in wound contraction. Ekperentia, 27: 549-550.
Greenlee, T. K., and D. Pike 1971 Studies of
tendon healing i n the rat: Remodelling of the
distal stump after severance. Plast. Reconstr.
Surg., 48: 260-270.
Imura, S., S. Tankaka and B. Takase 1975 Intracytoplasmic segment long spacing fibrils in
chondrosarcoma. J. Electron Microsc., 24: 8795.
Listgarten, M. A. 1973 Intracellular collagen
fibrils in the periodontal ligament of the mouse,
rat, hamster, guinea pig and rabbit. J. perio.
Res., 8: 335-342.
Luse, S., and R. Hutton 1964 A n electron microscopic study of the fate of collagen i n the postpartum rat uterus. Anat. Rec.. 148: 308.
Montfort, I . , and R. Perez-Tamayo 1975 The
distribution of collagenase in the rat uterus during postpartum involution. Connect. Tissue Res.,
3: 245-252.
Parakkal, P. F. 1969 Involvement of macrophages in collagen resorption. J. Cell Biol., 41 ;
Peppler, R. D. 1975 Reduction in eggs ovulated
in rats with a copper intrauterine device. Contraception, 12: 327-332.
PCrez-Tamayo, R. 1970 Collagen resorption in
carrageenin granulomas. 11. Ultrastructure of
collagen resorption. Lab. Invest., 22: 142-159.
Schwarz, W., and F. H. Gulden 1967 Elektron-
enmikroskopische Untersuchungen des Kollagenabbaus i n Uterus der Ratte nach der Schwangerschaft. Z. Zellforsch., 83: 416426.
Tansey, T. R., and H. A. Padykula 1976 Cellular responses to experimental inhibition of resorption in the postpartum rat uterus. Anat.
Rec., 184: 544-545.
Tang Kao, K., D. M. Hilker and T. H. McGavack
1961 Connective tissue V. Comparison of synthesis and turnover of collagen and elastin in
tissues of rat at several ages. Proc. Soc. Exp.
Biol. Med., 106: 335-338.
Ten Cate, A. R. 1972 Morphological studies of
fibrocytes in connective tissue undergoing rapid
remodelling. J. Anat., 112: 401-414.
Ten Cate, A. R., and D. A. Deporter 1974 The
role of the fibroblast in collagen turnover i n the
hnctioning periodontal ligament of the mouse.
Archs. oral Biol., 19: 339-340.
Ten Cate, A. R., and E. Freeman 1974 Collagen
remodelling by fibroblasts i n wound repair. Preliminary observations. Anat. Rec., 279: 543-546.
Ten Cate, A. R., and S. Syrbu 1974 A relationship between alkaline phosphatase activity and
the phagocytosis and degradation of collagen by
the fibroblast. J. Anat., 1 1 7: 351-359.
Intracellular collagen fibril (arrow) in stromal cell with morphological
features of a macrophage. The cell is adjacent to the basal lamina (EL)
of the uterine epithelium (E).Three-month control animal. X 27,200.
Intracellular collagen fibril within a phagosome (arrow). Three-month
control animal. X 25,500.
Intracellular collagen fibrils within a phagolysosome (P). Three-month
control animal. X 34.500.
Robert F . Dyer and Richard D . Peppler
Collagen fibrils located peripherally within a phagolysosome (P). Three
month copper IUD. X 45,750.
Collagen fibrils within a phagolysosome, considered to be in a terminal
phase of degradation. Periodicity is most clearly seen at the arrow.
Three-month copper IUD. X 82,000.
A single macrophage type cell associated with collagen fibrils in different stages of degradation. Fibrils at arrow A may be in the process of
being phagocytosed. Fibrils at arrow B appear to be within the cytoplasm, and arrow C indicates fibrils within a phagolysosome. Threemonth control. X 21,900.
Robert F . Dyer and Richard D. Peppler
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uterus, containing, nonpregnant, intracellular, iud, rat, collagen
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