lntracellular Collagen in the Nonpregnant and IUD-containing Rat Uterus ROBERT F. DYER AND RICHARD D PEPPLERZ Departments of Anatomy and Obstetrics and Gynccology, Louisiana State Unzversity Medical Center, New Orleans, Louzstana 70112 ABSTRACT 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, ANAT. 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. MATERIALS AND METHODS 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 1 242 ROBERT F. DYER AND RICHARD D. PEPPLER 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 together. 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. RESULTS 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 stroma. 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). DISCUSSION 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- INTRACELLULAR COLLAGEN 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 243 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). LITERATURE CITED 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 ; 345-354. 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- 244 ROBERT F. DYER AND RICHARD D. PEPPLER 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. PLATE 1 EXPLANATION OF FIGURES 1 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. 2 Intracellular collagen fibril within a phagosome (arrow). Three-month control animal. X 25,500. 3 Intracellular collagen fibrils within a phagolysosome (P). Three-month control animal. X 34.500. INTRACELLULAR COLLAGEN Robert F . Dyer and Richard D . Peppler PLATE 1 245 PLATE 2 EXPLANATION OF FIGURES 246 4 Collagen fibrils located peripherally within a phagolysosome (P). Three month copper IUD. X 45,750. 5 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. 6 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. INTRACELLULAR COLLAGEN Robert F . Dyer and Richard D. Peppler PLATE 2 247
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