1 Vincent Memorial Obstetrics and Gynecology Service and 2 Wellman Laboratories of Photomedicine, Massachusetts General Hospital and Harvard Medical School, Fruit Street, Boston, MA 02114, USA
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Abstract |
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Key words: animal model/complement C3/cytokeratin/endometriosis/SCID mouse
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Introduction |
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Since menstrual shedding is a requirement for the spontaneous development of the disease, endometriosis is primarily a condition of primates. The low prevalence of spontaneous endometriosis in the monkey and the high costs of animal handling, however, limit the use of this model in endometriosis research (MacKenzie and Casey, 1975; Fanton and Hubbard, 1983
). Oestrous animals, on the other hand, do not shed their endometrial elements and therefore do not develop the disease spontaneously. The use of these animals as experimental models thus entails the surgical transplantation of autologous endometrial tissue to ectopic locations in the abdomen. This technique has been described in various laboratory animals, including rabbits, rats and monkeys (Schenken and Asch, 1980
; Vernon and Wilson, 1984
; Dunselman et al., 1989
; Schenken et al., 1991
; Sharpe et al., 1991
; D'Hooghe et al., 1995
). These models, however, possess many limitations because significant phylogenetic and biochemical differences exist between human and animal endometria. Considerable species differences also make histological observations in these animals at times inapplicable to the human endometrium. Furthermore, biological responses are not always the same, and some proteins induced by oestradiol in the rodent endometrium are augmented by progesterone in humans.
The ability to transplant human endometrium into animals provides an attractive approach with several advantages over existing animal models, but remains limited by immunological graft rejection reactions. Immunocompromised mice offer the unique opportunity to study the behaviour and biochemical expression of human endometrium when transplanted into ectopic locations in vivo. Severe combined immunodeficient (SCID) mice possess a combined congenital deficiency in T- and B-lymphocyte function, and have recently been described to host various heterotransplants successfully (Phillips et al., 1989). Serially passed human tissues were shown to retain their morphological as well as their biochemical characteristics (Visfeldt et al., 1972
; Shimosato et al., 1976
; Stiles et al., 1976
; Kim et al., 1978
; Satyaswaroop et al., 1983
).
The aim of the present study was to validate the suitability of the SCID mouse as an experimental model for endometriosis, by defining some of the morphological, histological and functional features of human endometrial implants and by characterizing specific biochemical properties of these implants.
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Materials and methods |
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Collection and processing of tissues
After approval of the Massachusetts General Hospital Committee on Human Studies on the use of human residual tissue, fresh human late secretory endometrial tissue (days 812 from ovulation) was obtained from premenopausal women undergoing endometrial biopsy or elective hysterectomy for benign gynaecological disease.
Endometrial tissues from the luteal phase of the menstrual cycle were collected in cold sterile Dulbecco's phosphate-buffered saline (PBS) (Sigma, St Louis, MO, USA), washed twice to remove cellular debris, then suspended as coarse fragments in PBS with 200 IU/ml of penicillin (Sigma) and 200 µg/ml of streptomycin (Sigma). Endometrial tissue was then loaded in 3 ml syringes, and standardized with respect to volume and weight. A volume of 0.6 ml of human secretory endometrium, i.e. the equivalent of 0.8 g of wet tissue, was then injected blindly using a 16-gauge needle into the peritoneal cavity of SCID mice on the ventral midline within 60 min of procurement. Endometrial dating of preinoculated secretory samples was performed using the criteria of Noyes (Noyes et al., 1950).
Hormonal therapy was initiated at the time of injection, and at 5 day intervals thereafter. Mice were divided into two groups to receive one of two hormonal regimens: (a) 17-ß-oestradiol-3-benzoate (30 µg/kg i.m.; FisherBiotech, Fair Lawn, NJ, USA) or (b) a combination of oestradiol/progesterone (10 mg/kg i.m.; United Research Laboratories, Bensalem, PA, USA).
Implanting tissue
Fourteen days following injection, the animals were killed using an overdose of pentobarbital sodium (Sigma). Implanted endometrial lesions were then identified, resected, and fixed in 10% neutral buffered formalin and embedded in paraffin. Microtome tissue sections, 4 µm thick, were placed on 1% poly-D-lysine coated glass slides (Superfrost, Fisher Scientific, Pittsburgh, PA, USA), and saved for later histological, immunohistochemical and in-situ hybridization analysis.
Immunohistochemistry
Paraffin-embedded tissue sections were subjected to deparaffinization and rehydration prior to staining by preincubation for 20 min with 0.1% trypsin in PBS at 37°C to expose antigenic sites. Endogenous peroxidase activity was then exhausted by incubation with 0.3% H2O2 in 96% methanol for 5 min. After rinsing in PBS, non-specific binding was blocked by incubation with normal goat serum (1/100 dilution in PBS) for 30 min. Sections were then kept overnight at 4°C with a 1/125 dilution of human pan-cytokeratin antibody (rabbit polyclonal; BioGenex, San Ramon, CA, USA). They were washed with PBS, then exposed sequentially to biotinylated secondary antibody (30 min), and avidin-biotinylated horseradish peroxidase complex (60 min) at room temperature (Vectastain Elite ABC detection system; Vector Laboratories, Burlingame, CA, USA). Sections were then incubated with the peroxidase substrate 3,3' diaminobenzidine for 5 min to yield a brown reaction product, counterstained with haematoxylin for 5 min, rinsed in PBS and dipped in a lithium carbonate solution for 30 s. They were then dehydrated in ascending ethanol concentrations (70, 80, 95 and 100%) for 2 min each and coverslipped with Permount out of xylenes.
In-situ hybridization
Paraffin-embedded tissue sections were prepared for in-situ hybridization using RNase-free techniques as follows: sections were deparaffinized in xylene, treated with proteinase K, and air-dried for hybridization.
Human liver complement 3 (C3) cDNA clone pHLC3.11 was obtained from ATCC (Rockville, MD, USA). A restriction digestion with Ava1 and Hind III yielded a 736 bp fragment representing sequence 19442680 of the 4.3 kb clone (De Bruijn and Fey, 1985). This fragment was purified by agarose gel electrophoresis and subcloned into the multiple cloning site of the pGEM-4Z vector according to the manufacturer's instructions (Promega, Madison, WI, USA). The pGEM-4Z subclone was linearized with Ava1 or Hind III providing templates for antisense and sense riboprobes. 35S-Labelled C3 riboprobes were generated by transcription from T7 and SP6 promoters respectively using the Riboprobe Gemini II Core System according to the manufacturer's instructions (Promega). The antisense probe was used to localize expression of endometrial C3, and the sense probe was used for control hybridization. Human liver sections were used as positive controls.
In-situ hybridization was carried out as follows: after prehybridization with cold buffer each tissue section was covered with 200 µl of hybridization buffer containing 20 00040 000 cpm/µl of sense or antisense probe and incubated overnight at 55°C in a humid chamber. The next morning, RNase A treatment (20 µg/ml) for 30 min at 37°C followed to reduce non-specific binding. After successive washing in a shaking bath, sections were dehydrated in ascending ethanol concentrations (50, 70, 90, 95, 100%) containing 300 mmol/l ammonium acetate, and then air dried. In a dark room, they were coated with a 1:1 dilution of NTB-2 photographic emulsion (Eastman Kodak, Rochester, NY, USA) and exposed at 4°C for 21 days. After developing (Kodak D19) and fixation (Kodak Rapid Fix), slides were counterstained with haematoxylin, dehydrated in an ascending alcohol gradient and coverslipped with Permount out of xylenes.
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Results |
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Histological analysis
Microscopically, haematoxylineosin staining of implant sections invariably demonstrated the presence of endometrial acinar glands in a mixed background of stromal and inflammatory cells. Implants from oestradiol-treated mice showed a variable number of small acinar structures with flat epithelial cells, surrounded by a densely cellular stroma (Figure 1a). Evidence of pseudostratification of nuclei was also noted. Tissue sections from oestradiol/progesterone-treated mice showed significant dilatation of acinar structures that looked tortuous and fluid-filled, reflecting the secretory nature of the glandular tissue (Figure 1b
). Glandular epithelial cells were more cuboidal with clear cytoplasm and occasional distinct vacuoles. Evidence of stromal decidualization was also seen. No mitotic figures were observed in any section.
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Discussion |
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Information on the clinical presentation of endometriosis in humans is widely available. However, little is known about the pathogenic processes leading to the development and perpetuation of the disease. By the time it is brought to medical attention, endometriosis is already well established. This fact decreases the opportunity to study the disease at early stages of its inception. Ethical considerations, furthermore, limit the ability to investigate the disease in humans in a well-designed experimental context. In view of these limitations, an alternate experimental animal model of endometriosis becomes a necessity. Since phylogenetic differences between species could significantly limit our understanding of the disease in humans, the idea of implanting human endometrium into host peritoneal surfaces is very attractive and relevant. Immunocompromised animals do not possess the ability to reject foreign tissue and therefore appear to be ideal candidates for such studies.
SCID mice offer the opportunity to create a humanized animal model for the study of experimental endometriosis, and help to bridge the phylogenetic gap that exists with autologous implants in other animal models. Benign human endometrium was reported to implant successfully in the subcutaneous tissue of these mice 10 weeks following injection (Bergqvist et al., 1985; Zaino et al., 1985
; Aoki et al., 1994
). The growth of human endometrium was found to be unaffected by the oestrous cycle stage of the animal (Aoki et al., 1994
). In the present experiment, the hormonal stimulation was induced thus rendering the oestrous stage of the animal irrelevant to the success of the implantation process. Although subcutaneous implants can provide more visual information on the growth and response of endometrial implants to environmental manipulation, the peritoneum represents the most natural host for the growth of these implants. Although Zamah (Zamah et al., 1984
) showed that endometrial tissue can implant successfully on the peritoneum of nude mice, we demonstrated a higher success rate of implantation of 96.5% in SCID mice. We also found that the most common location of implantation was the pelvis, the most dependent part of the animal, thus resembling closely the site of occurrence of the human disease.
In the present study, the administration of progesterone supplementation in vivo was associated with multiple peritoneal implantation sites and with significantly larger implants. Acinar glands became tortuous and dilated, evidence of conservation of functional responsiveness to hormonal manipulation. The presence of fluid-filled glandular lumens very likely represents preservation of the secretory capacity of these implants. However, these histological changes in response to steroids appeared to be atypical when compared to eutopic endometrium. Although endometriosis has been shown to be responsive to hormonal stimulation, recent biochemical, ultrastructural, and autoradiographic studies suggest that this response is indeed incomplete (Lessey et al., 1989). The findings of our study suggest that endometrial tissue assumes histological and functional features similar to endometriosis when transferred to ectopic locations.
Cytokeratins constitute a class of intermediate filaments present in the cytoplasm of human epithelial cells (Winter et al., 1980). Patterns of expression change with altered microenvironmental conditions, such as disease states (Cintorino et al., 1988
). Studies have shown that cytokeratin subtypes are present in the epithelial cells of the human endometrium and absent in stromal elements (Nisolle et al., 1995
). The pan-cytokeratin antibody used in this study was species-specific to humans. It stained implanted human glandular epithelium and failed to stain murine epithelium. This indicates that the human endometrium conserves specific structural cellular patterns throughout the complex process of implantation.
The third component of C3 is a glycoprotein that plays an important role in the humoral immune response in the body. A possible role in endometrial physiology and in the pathophysiology of endometriosis has been proposed (Weed and Arquembourg, 1980; Isaacson et al., 1989
, 1990
). In this study, we examined the ability of implanted endometrium to produce C3. The expression of C3 m-RNA by endometrial implants suggests that these ectopic endometrial implants were immunologically responsive to changes in the hosting environment. The presence of stronger hybridization signals in progesterone-treated implants denies the response described in rodents (Satyaswaroop et al., 1983
), and suggests that ectopic endometrial tissues also retained their human pattern of genetic control despite transplantation into a host with different phylar characteristics.
The SCID mouse model of endometriosis offers several advantages over other existing animal models. It provides the opportunity to study transformational and adaptational changes occurring in the human endometrium when implanted onto ectopic host sites. Monitoring of these changes at very early stages of implantation yields valuable information about the pathophysiology of endometriosis otherwise inaccessible to clinical studies. Moreover, the study of these events when endometrial tissues are obtained from women with endometriosis helps evaluate the intrinsic potential of the endometrium to invade the hosting site, thus giving new insights into the theory of Sampson (Sampson, 1927). The SCID mouse model can also be useful to evaluate the potential of the human endometrium to implant at different phases of the menstrual cycle. This can yield important information on particular characteristics of the endometrium that enhance its ability to implant at ectopic sites. Since it can be manipulated and optimized for various research purposes, this animal model can be utilized to assist in planning proposed treatment strategies. Other advantages of the SCID mouse include small size, low cost, and easy handling.
The main limitation of this animal model, however, is its inability to duplicate the immune changes occurring at the implantation site in humans. Another well-recognized difficulty in handling an immunodeficient mouse is its short life span when manipulated under conventional conditions, a problem partially preventable by the use of barrier facilities.
We conclude that the SCID mouse represents an attractive animal model for the study of endometriosis, by its ability to host human endometrial tissue in ectopic peritoneal locations efficiently and reproducibly. Endometrial implants retained histological characteristics, as well as functional responsiveness to hormonal stimulation. They also conserved specific aspects of their structural and biochemical composition, as well as secretory and immunochemical properties. This model thus offers the unique opportunity to study the transformational changes of eutopic human endometrial tissue when implanted at ectopic locations. It is also useful to assess the effects of pharmacological and hormonal modulations on endometriosis without the confounding effects of phylogenetic differences.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 22, 1999; accepted on September 7, 1999.