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Articles |
a Center for Animal Biotechnology and Genomics, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, and Department of Animal Science, Texas A&M University, College Station, Texas 77843
b Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843
c Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View, Texas 77446
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actin staining. Western blot analyses of cell extracts revealed the presence of signal transducers and activators of transcription (STAT) proteins 1, 2, and 3. In the LE cells, interferon tau (IFN
) induced nuclear translocation of STAT proteins 1 and 2 and up-regulated several IFN-inducible genes, including STATs 1, 2, and 3 and ubiquitin cross-reactive protein (UCRP/ISG17). In the LE cell line, IFN regulatory factor one was transiently up-regulated and then down-regulated by IFN. Immunostaining revealed the presence of nuclear estrogen receptor and progesterone receptor in all cell lines. These ovine endometrial cell lines provide useful in vitro model systems for the study of hormone and cytokine action, signal transduction pathways, cell-cell interactions, and gene expression in specific cell types of the ovine endometrium.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In ruminants, regular estrous cycles, as well as establishment and maintenance of pregnancy, require integration of both endocrine and paracrine signals from the ovary, conceptus, and uterus [8]. Endometrial production of luteolytic prostaglandin F2 (PGF2) in cyclic ewes is regulated by complex interactions between estrogen, progesterone, oxytocin, and their respective receptors in the uterus [8–11]. However, in pregnant ewes, pulsatile production of luteolytic PGF2 by the endometrial LE and superficial GE is abrogated by interferon tau (IFN) secreted from the conceptus trophectoderm [8]. In pregnant ewes, IFN suppresses epithelial estrogen receptor (ER) and oxytocin receptor (OTR) gene expression to interrupt the luteolytic mechanism and provides the signal for pregnancy recognition [8, 12, 13]. It appears that IFN acts directly on the LE and superficial GE during pregnancy to sequentially induce interferon regulatory factor one (IRF-1) and then IRF-2 gene expression, which, according to a current hypothesis, suppresses transcription of the ER and perhaps OTR genes [14]. In addition to its role in pregnancy recognition, IFN increases uterine expression of 2',5'-oligoadenylate synthetase [15], ß2-microglobulin [16], Mx protein [17], granulocyte chemotactic protein 2 [18], and ubiquitin cross-reactive protein (UCRP) [19, 20]. Recent evidence indicates that effects of IFN are not limited to the endometrial epithelia, because UCRP and Mx mRNA can be detected throughout the entire ovine uterine wall, including stromal and myometrial cell types, between Days 11 and 19 of pregnancy [17, 21, 22].
Investigations into the cellular and molecular mechanisms whereby endocrine and paracrine hormones affect endometrial function in ruminants have been hampered by the lack of cell lines that retain a differentiated phenotype. Normal primary endometrial cells are not ideal for long-term studies, because they undergo some de-differentiation in culture (e.g., loss of hormone and growth factor/cytokine responsiveness) and have an inherently short replicative life span before senescence. A defective retroviral construct (LXSN-16E6E7) that stably expresses the human papillomavirus (HPV) type 16 E6 and E7 proteins [23] has been used to immortalize a number of epithelial cell types, including human ectocervical, endocervical, and vaginal epithelium [24] and ovarian epithelium [25]. Expression of HPV E6 and E7 viral oncogenes increases the proliferative capabilities of epithelial cells, but the resulting immortalized cell lines are not completely transformed. HPV-immortalized cell lines are incapable of anchorage-independent growth and are not tumorigenic in nude mice, and they retain the normal karyotypes and stable phenotypes of their cell type of origin [24, 25]. Therefore, the objective of the present study was to establish HPV 16 E6E7-immortalized endometrial LE, GE, and ST cell lines from the ovine uterus that retain endocrine and paracrine signaling pathways characteristic of their in vivo and/or primary cell counterparts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature Western range ewes of primarily Rambouillet breeding were observed daily for estrous behavior in the presence of vasectomized rams. After two estrous cycles of normal duration (16–18 days), ewes were assigned on Day 0 (estrus) to be hysterectomized on Day 5 of the estrous cycle for isolation of uterine LE, GE, and ST cells. All experimental and surgical procedures involving animals were approved by the Agricultural Animal Care and Use Committee of Texas A&M University (Animal Use Protocols 7–286 and AG-239).
Establishment of Primary Cell Lines
Endometrial LE, GE, and ST cells were isolated as previously described [26] with modifications. Immediately after hysterectomy, the uterus was separated into segments. One half of one uterine horn adjacent to the oviduct was used for isolation of LE cells, and the remainder of the uterus was used for isolation of GE and ST. The lumen of the largest uterine segment was flushed twice with warm (37°C) Ca2+/Mg2+-free Hanks' Balanced Salt Solution (HBSS; Sigma Chemical Co., St. Louis, MO), filled with approximately 20 ml of HBSS containing pancreatin (2.5 mg/ml; Gibco-BRL, Grand Island, NY) and dispase II (4.8 mg/ml; Boehringer Mannheim, Indianapolis, IN), and incubated at 37°C for 1 h. Enzyme was then gently removed from the uterine horn and discarded. The uterus was refilled with HBSS and incubated at room temperature for 15 min with gentle massage to loosen LE sheets. The wash was then removed from the uterine horn, and the horn was washed repeatedly until a reduced yield of LE sheets was observed. The washes were then pooled. The LE cell sheets were dispersed using a reduced-bore pipette, and LE cells were recovered by centrifugation (500 x g for 10 min). The resulting LE cell population was resuspended in complete culture medium, plated, and expanded in 75-cm tissue culture flasks. Complete culture medium was Dulbecco's modified Eagle's medium with F12 salts (DMEM/F12; Sigma), pH 7.4, containing 10% (v:v) fetal bovine serum (FBS; Gibco-BRL) and penicillin/streptomycin/amphotericin B solution (100 IU/ml, 0.1 mg/ml, 0.25 µg/ml; Gibco-BRL).
To isolate uterine GE cells, endometrium was physically dissected from the remaining segment of the uterine horn, minced, and placed into 40 ml of HBSS containing DNase I (200 U/ml; Boehringer-Mannheim) and type III collagenase (1 mg/ml; Boehringer-Mannheim). Endometrium was incubated at 37°C in a sterile 50-ml tube with occasional agitation for approximately 1 h, at the end of which time a cloudy supernatant was evident. The cellular homogenate was allowed to sediment at unit gravity, and enriched GE cells were collected from the interface between the tissue pellet and supernatant. The recovered GE cells were centrifuged (500 x g for 10 min), resuspended in complete culture medium, and expanded in 75-cm tissue culture flasks.
To isolate ST cells, the supernatant and tissue pellet were centrifuged at 500 x g for 10 min. Resulting cells and tissue fragments were then separately washed with HBSS; resuspended in complete culture medium, plated, expanded in 75-cm tissue culture flasks; and cultured for 48 h at 37°C and 5% CO2. Stromal cells were then removed from culture flasks with 0.1% (w:v) trypsin in PBS containing 0.53 mM EDTA (pH 7.6) and seeded into new tissue culture flasks.
HPV 16 E6E7 Immortalization
PA-317 cells were purchased from the American Tissue Type Culture Collection (Manassas, VA). Virus was harvested from PA-317 cells by addition of fresh culture medium to confluent 75-cm tissue culture flasks; this was followed by 16 h of culture, removal of medium, and filtration through a 0.45-µm low protein binding filter (Millipore, Bedford, MA) to remove cells and debris [27]. Viral supernatant (1 ml; containing LXSN-16E6E7) was combined with 3 ml of serum-free DMEM/F12 medium containing polybrene (4 µg/ml; Sigma) and overlaid onto third-passage primary endometrial LE, GE, and ST cells that had been grown to 60% confluence in 75-cm tissue culture flasks and derived from cultures of one uterus obtained from a Day 5 cyclic ewe. After 3-h incubation at 37°C under 5% CO2, 5 ml of serum-free DMEM/F12 containing 4 µg/ml polybrene was added to the flasks, and cells were further incubated for 4 h at 37°C and 5% CO2. Viral supernatant was then removed; cells were washed twice with HBSS, and complete culture medium was added to flasks. After 48 h, culture medium was removed, and cells having integrated the vector were selected by resistance to the neomycin analogue G418 (Gibco-BRL; 800 µg/ml for LE and GE, 600 µg/ml for ST) in complete culture medium. Selection continued for 7 days, and surviving cells were subsequently cultured with complete culture medium containing 0.1 mg/ml G418 for general cell maintenance [25]. The G418-resistant cell populations were subcultured for more than 30 passages, and immortalized cells were cryopreserved each passage.
Antibodies for Immunofluorescence and Western Blot Analyses
Antibodies to cytokeratin (6909), vimentin (V-6630), smooth muscle -actin (A-2547), and desmin (D-1033) were purchased from Sigma. Polyclonal rabbit anti-human ER antibody (18–0174) was from Zymed Laboratories, Inc. (San Francisco, CA), and mouse monoclonal antibody to human progesterone receptor (MS-192-P1) was from Neomarkers (Fremont, CA). Mouse monoclonal antibodies to STAT2 (G16920) and STAT3 (S21320) were from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibody to human STAT1 (sc-476) and IRF-1 (sc-497) was from Santa Cruz Biotechnology, (Santa Cruz, CA). Rabbit antiserum to human UCRP was kindly donated by Dr. Ernest Knight Jr. (E.I. du Pont de Nemours & Company, Wilmington, DE) [28].
Immunofluorescence Microscopy Analysis
Immortalized ovine endometrial LE, GE, and ST cell monolayer cultures were grown on LabTek 4-well chamber slides (Nunc, Naperville, IL). For detection of cytokeratin, vimentin, smooth muscle -actin, desmin, and ER, cells were immediately fixed in -20°C methanol for 10 min. For detection of STAT1 and STAT2, cells were incubated in serum-free DMEM/F12 for 45 min before being treated with recombinant ovine IFN (104 antiviral U/ml) for 0, 10, 30, or 60 min and then fixed in -20°C methanol. For detection of progesterone receptor (PR), cells were immediately fixed in 2% paraformaldehyde in 0.02 M PBS for 10 min, washed in 0.02 M PBS two times for 5 min each, and then permeabilized in 1% Triton X-100 in 0.02 M PBS for 10 min. All slides were then blocked in antibody dilution buffer (two parts 0.02 M PBS, 1.0% BSA, 0.3% Tween 20, pH 8, and one part glycerol) containing 5% normal serum (from the species in which the respective secondary antibody was raised) for 1 h at room temperature. After a quick rinse in 0.02 M PBS containing 0.3% Tween 20 (PBS), slides were incubated overnight at 4°C with primary antibody.
After three rinses in PBS for 10 min each, immunoreactive proteins were detected by one of two methods: 1) for detection of cytokeratin, vimentin, smooth muscle -actin, desmin, STAT1, and STAT2, slides were incubated with fluorescein-conjugated secondary antibody (goat anti-rabbit IgG, Zymed; or rabbit anti-mouse IgG; Sigma) for 1 h at room temperature and again washed in PBS (3 times, 10 min each); 2) for detection of ER and PR, slides were incubated with biotinylated secondary antibody (sheep anti-mouse IgG or donkey anti-rabbit IgG; Amersham Life Sciences, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at room temperature, washed in PBS (3 times, 10 min each), incubated in fluorescein-conjugated streptavidin (Amersham) for 1 h at room temperature, and again washed in PBS (3 times, 10 min each). All slides were then overlaid with a coverglass and Prolong antifade mounting reagent (Molecular Probes, Eugene, OR), viewed with a Zeiss Photomicroscope III (Zeiss, Thornwood, NY) equipped with a filter set for fluorescein, and photographed using T-MAX 3200 film (Eastman Kodak, Rochester, NY).
Western Blot Analysis
Immortalized ovine endometrial LE, GE, and ST cell monolayer cultures were grown to 90% confluence on 150-mm tissue culture plates (Nunc). Cells were then treated with recombinant ovine (ro) IFN (104 antiviral U/ml) for 0, 3, 6, 12, 24, or 48 h, rinsed with ice-cold PBS, collected into 1.5 ml ice-cold PBS by scraping, and pelleted by centrifugation. The IPH lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1 mM PMSF) (250 µl) was then added to cell pellets with vortexing, and cells were incubated for 20 min on ice. Cellular debris was pelleted by centrifugation (12 000 x g for 10 min).
Concentrations of protein in cellular extracts were determined using a Bradford protein assay (Bio-Rad, Hercules, CA) with BSA as the standard. Proteins in extracts (20 µg) were denatured in Laemmli buffer, separated on 10% (total monomer) SDS-PAGE gels, transferred to nitrocellulose, and immunoprobed as previously described [29]. Blots were incubated with primary antibodies or control serum or IgG at 5 µg/ml overnight at 4°C. Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham) and Kodak X-OMAT AR Film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The uterus from a Day 5 cyclic ewe was used on the basis of observations that cells isolated from uteri on Day 12 or 15 of the cycle exhibit slow growth rates and are difficult to propagate (unpublished results). Transduction of primary ovine endometrial cells yielded one G418-resistant colony for the LE and stroma and two colonies for the GE. The immortalized ovine endometrial LE, GE, and ST cell lines were maintained in continuous culture for greater than 40 population doublings without signs of senescence, whereas primary cell cultures reached senescence within 7 to 10 passages. The immortalized LE and GE cells had increased growth rates, with population doubling times of about 30 h compared to about 72 h for primary cultures. Both LE and GE cells reached higher cell densities than primary cells and had a distinctive cobblestone morphology (Fig. 1). The LE cells demonstrated looser colonies that expanded to all available space before packing tightly into monolayers. In contrast, a tight sheet-like pattern of growth, which was not inhibited at confluency, was observed for GE cells. Immortalized ST cells, however, exhibited a stellate shape (Fig. 1) that became more fibroblast-like upon crowding. These cells did not grow more rapidly (passage every 5–7 days) than primary cell cultures, and growth was inhibited by cell splitting ratios exceeding 1:2.
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Analysis of intermediate filament protein expression in immortalized cells was performed on cells between passages 20 and 25 (Fig. 1). Both immortalized LE and GE cells were stained by the epithelium-specific cytokeratin antibody, whereas immortalized ST cells were not immunoreactive to this antibody. Immortalized fibroblast-like ST cells exhibited vimentin staining (Fig. 1), as did immortalized LE and GE cells (data not shown). Immortalized LE, GE, and ST cell lines exhibited no immunoreactivity to smooth muscle -actin (Fig. 1).
Functional Characterization of Immortalized Endometrial Cell Lines
Estrogen and progesterone receptors Both ER and PR were detected in monolayers of LE, GE, and ST cells (Fig. 2). The ER staining was uniformly intense in each of the cell types. PR staining was less intense than the ER staining. As expected, the majority of immunoreactive ER and PR protein was detected in the nucleus.
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STAT proteins and their nuclear translocation
Since ovine uterine LE cells in vivo express receptors for IFN, evidence for IFN-mediated signal transduction in LE cells was examined. Western blot analysis of LE cells revealed the expression of immunoreactive STATs 1, 2, and 3 proteins in cytosolic extracts of immortalized LE cells (Fig. 3). The STAT 1 (
85 kDa), STAT 2 (115 kDa), and STAT 3 (90 kDa) proteins were expressed constitutively by LE cells. Treatment of these cells with roIFN increased expression of STATs 1, 2, and 3 proteins. The controls, in which mouse or rabbit IgG replaced antibodies to STAT proteins, showed no cross-reacting 85-, 115-, or 90-kDa proteins. For STAT 2, the 60-kDa and 40-kDa immunoreactive bands probably represent degradation products due to the short half-life of the STAT proteins and on the basis of the negative control.
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Immunocytochemical analysis of STAT 1 and STAT 2 proteins in LE cells indicated that the expression of these proteins was predominantly cytoplasmic (Fig. 4). However, upon stimulation of the cells with roIFN (104 AVU/ml), translocation of both STAT 1 and STAT 2 proteins from the cytoplasm to the nucleus was detected as early as 30 min and was maximal at 60 min.
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IFN modulation of IRF-1 and UCRP expression
Immunoreactive IRF-1 was detected in immortalized ovine LE cells by Western blot analysis (Fig. 5). Stimulation of the ovine LE cells with roIFN for 3, 6, or 12 h transiently increased IRF-1 protein. However, roIFN treatment for 24 h or 48 h decreased IRF-1 expression. The controls, in which normal rabbit IgG replaced polyclonal anti-IRF-1 serum as the primary antibody, showed no cross-reacting IRF-1 protein.
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Immunoreactive UCRP (17 kDa) and ubiquitin (8 kDa) proteins were detected in immortalized ovine endometrial LE, GE, and ST cells (Fig. 6). The rabbit antiserum to human UCRP [28] detects both proteins. The expression of UCRP protein increased in all three endometrial cell lines in response to treatment with roIFN. In contrast, expression of ubiquitin protein remained constant and was unaffected by roIFN. The negative controls, in which normal rabbit serum replaced polyclonal anti-human UCRP serum as the primary antibody, showed no cross-reacting proteins corresponding to the 17-kDa UCRP and 8-kDa ubiquitin proteins (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ovine endometrial cell lines should provide a valuable model for study of the actions of hormones, cytokines, and growth factors. Results of the present study indicate that the LE cell line is particularly useful for the study of IFN signal transduction. Western blot analyses indicated that the LE cell line expressed STATs 1, 2, and 3. The LE cell line responds to IFN with translocation of STATs 1 and 2 to the nucleus and increased levels of STATs 1, 2, and 3 proteins. This agrees with results from other cell models wherein STATs 1, 2 and 3 are regulated by IFN-, a member of the type I IFN family [33].
Type I IFNs also regulate expression of IRF-1 and UCRP. Using an in vivo uterine catheterization model, Spencer et al. [14] found that intrauterine administration of IFN up-regulated expression of IRF-1 protein at 12 and 24 h after initial injection in LE and superficial GE of the ovine uterus. However, IRF-1 protein could not be detected at 48 h to 120 h after IFN treatment. In the present study, treatment of the LE cell line with IFN appeared to transiently increase IRF-1 protein levels in cells between 0 and 12 h, and levels decreased thereafter. The three immunoreactive proteins found at only the 3-, 6-, and 12-h time points most likely represent increased levels of IRF-1 protein that is degraded due to the short, 30-min half-life of the protein [34]. These in vitro results are similar to those observed in vivo. The acceleration in timing of response to IFN in immortal LE cells may be due to the low level of constitutive expression of IRF-1, as well as STATs 1 and 2, in these cells. Because the LE in vivo does not appear to express IRF-1 protein constitutively, up-regulation of factors involved in signal transduction is likely required.
During the period of pregnancy recognition in sheep, UCRP mRNA is localized to the LE, stratum compactum layer of the ST, and superficial GE on Day 13. Moreover, expression extends into the deep GE, stratum spongiosum of the ST, and myometrium on Days 15 through 19 [21]. In the bovine uterus, UCRP mRNA has also been localized to both endometrial GE and ST during pregnancy [20]. The UCRP is up-regulated by IFN in bovine endometrial explants and the ovine uterus [19, 20, 22]. Thus, it is noteworthy that all of the immortalized ovine endometrial cell lines are responsive to IFN. Treatment of the LE, GE, and ST cell lines with IFN up-regulated UCRP protein expression while levels of ubiquitin protein expression were unaffected. These results agree with previous reports on the differential effects of IFN in vivo on UCRP and ubiquitin expression in the ovine and bovine endometrium [20, 23].
Collectively, morphological and biochemical evidence indicates that the immortalized ovine endometrial cell lines described here will provide useful in vitro model systems for the study of hormone and/or cytokine action and gene expression in specific cell types of the ovine endometrium. Furthermore, these endometrial cell lines, along with an ovine immortalized myometrial cell line that is also being developed using similar procedures as described here (unpublished results), offer additional opportunities to dissect functional interactions between uterine cell types in vitro. In particular, experiments that address steroid hormone-directed and cytokine/growth factor-mediated epithelial-stromal-myometrial interactions may be addressed in coculture experiments using cells that maintain functional properties characteristic of their in vivo counterparts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 444 Kleberg Center, Texas A&M University, College Station, TX 77843–2471. FAX: 409 862 2662; tspencer{at}ansc.tamu.edu
Accepted: June 22, 1999.
Received: May 19, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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