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Reproductive Biology Program, Department of Animal Science (K.J.A., A.L.C., J.K.P., E.L.G.), and Department of Veterinary Science and Veterinary Diagnostic Laboratory (E.L.B., C.E.H.), University of Wyoming, Laramie, Wyoming 82071
Address all correspondence and requests for reprints to: Thomas R. Hansen, Reproductive Biology Program, Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071-3684. E-mail: thansen{at}uwyo.edu.
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Abstract |
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on d 18 of pregnancy. It was hypothesized here that cellular localization of ISG15 within endometrial cells might provide insight regarding function. Uteri were collected from cows (
21-d estrous cycle) on d 17–21/0 of the estrous cycle and pregnancy and d 23, 45, and 50 of pregnancy. Intracellular ISG15 and its conjugates were present on d 17 of pregnancy, peaked to highest levels from d 18 to 23 and then declined to low but detectable levels by d 45 (P < 0.05) based on Western blotting. ISG15 and its conjugates were not detected on d 50 of pregnancy or during the estrous cycle. Immunohistochemistry revealed that ISG15 was localized throughout the endometrium on d 18–23, with heaviest staining in the sublumenal stratum compactum and the glandular epithelium throughout the stratum spongiosum. By d 45 and 50, ISG15 was lightly stained only in the stratum compactum immediately beneath the lumenal epithelium. Using transmission electron microscopy and immunogold labeling, ISG15 was specifically localized to organelles and compartments of endometrial epithelial cells and stromal cells: nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum, and cell membrane. This specific localization in epithelial and stromal cells led to the conclusion that ISG15 has diverse intracellular functions. The sustained presence of conjugated ISG15 through d 50 of pregnancy might reflect stabilization of conjugated proteins in response to implantation and the development of the placenta. |
Introduction |
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The mechanism through which pregnancy is recognized and established is quite diverse across mammals. For example, chorionic gonadotropin is believed to be responsible for establishment of pregnancy in primates (1), whereas conceptus-derived interferon- (IFN) is responsible for establishment of pregnancy in ruminants (2, 3, 4). Although some of the primary signaling mechanisms vary among mammals, some changes in the uterus are universal in response to pregnancy. For example, some degree of adhesion and endometrial remodeling occurs during implantation of the conceptus into the uterine wall, regardless of species. Likewise, an apparent universal response to the embryo is up-regulation of the ubiquitin homolog, called ISG15, in endometrium from humans (5, 6), baboons (6), cows (7, 8, 9, 10, 11), sheep (12, 13, 14, 15), swine (16), and mice (17).
IFN is released by the ruminant conceptus and induces an interferon-stimulated gene (ISG) that encodes a 17-kDa uterine protein (ISG15; also called ISG17) (7, 11). ISG15 was originally named ubiquitin cross-reactive protein due to cross-reactivity with antibody against ubiquitin (7, 8, 9, 11, 18). Human and mouse ISG15 homologs have a mature molecular weight of 17,000 but have an apparent Mr of 15,000 on PAGE and so were named ISG15 (19, 20, 21, 22, 23, 24). ISG15 also has been shown to be up-regulated in mouse (17) and human (5, 6) endometrium in response to pregnancy. These ubiquitin homologs are induced by type 1 interferon and become conjugated to proteins in a manner that is similar, but distinct from, that described for ubiquitin (9, 23). IFN mRNA is expressed by the conceptus in the pregnant cow by d 14, peaks on d 17–18, and then declines from d 21–26 (25, 26). The expression of ISG15 mRNA in the uterus (18) parallels the pattern of IFN release during early pregnancy. ISG15 mRNA is localized to glandular epithelium, stroma, and myometrium (10, 12) with less localization to lumenal epithelium in the cow (10) and very limited or no localization to the lumenal epithelium in the ewe (12).
Use of an anti-boISG15 peptide antibody failed in immunohistochemical experiments. Thus, the first objective of the present experiments was to generate a monoclonal antibody that immunoreacted with boISG15 but not ubiquitin for immunohistochemical applications. The second objective was to use this monoclonal antibody to localize ISG15 in uterine cross-sections during pregnancy and the estrous cycle and quantitate this response to pregnancy using one-dimensional PAGE and Western blotting. The hypothesis that ubiquitin conjugates did not change across the same days of pregnancy or the estrous cycle was also tested. The final objective was to use the monoclonal anti-ISG15 antibody in immunogold labeling and electron microscopy to determine whether ISG15 was localized to specific organelles or compartments within the cells. It was hypothesized that specific subcellular localization of ISG15 might provide insights regarding function of this ubiquitin homolog in the endometrium during early pregnancy.
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Materials and Methods |
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The fusion procedure used was a modification of that first used by Kohler and Milstein (28). Lymphocytes in HBSS were transferred to a 50-ml conical tube containing 1 x 107 SP2/0-Ag14 myeloma cells (29). Cells were mixed together with gentle pipetting, and 30 ml HBSS were added. Cells were pelleted via centrifugation (1400 rpm, 10 min, 4 C). Supernatant was decanted, and cells were fused at 37 C by the addition of a 50% solution of polyethylene glycol (Sigma Chemical Co.) over a 1-min interval. Polyethylene glycol was diluted with the addition of 7 ml hybridoma medium (30) added over a 3-min interval to maintain viability of fused hybridoma cells. Cells were pelleted via centrifugation (1400 rpm, 10 min, 22 C) and resuspended in 30 ml hybridoma medium supplemented with 20% fetal bovine serum (FBS). The cell suspension was transferred to three flat-bottomed 96-well microtiter plates using a 10-ml pipette (2 drops/well), and cells were incubated (37 C, 5% CO2) for 24 h. Selection of hybridoma cells was initiated by adding 50 µl hypoxanthine aminopterin thymidine (HAT) medium (100 ml hybridoma medium supplemented with 20% FBS and 2 ml HAT; Sigma Chemical Co.). On alternate days, through d 11, 100 µl medium were removed from each well and replaced with 100 µl HAT medium to maintain selection. Every day thereafter, through d 23, 100 µl medium were removed and replaced with 100 µl hypoxanthine thymidine medium (100 ml hybridoma medium supplemented with 20% FBS and 2 ml hypoxanthine thymidine; Sigma Chemical Co.). By d 23, selection was complete, and medium was exchanged daily with hybridoma medium supplemented with 20% FBS.
Fifty microliters of cell culture supernatant from each well were tested for the presence of rboISG15-positive antibodies via indirect ELISA as described below. Thirty-two positive clones were transferred to 24-well plates. Of these, 14 continued to produce antibodies. One hundred microliters of the five strongest polyclones were transferred back to 96-well plates and used for limiting dilutions to identify monoclonal hybridomas. Polyclones were placed in the top left corner of the plate and diluted 1:2 horizontally and then 1:2 vertically. ELISA was again used to identify antibody-producing monoclones 2 wk after making limiting dilutions. Twelve monoclones were selected for expansion in 24-well plates. Of these, seven continued to produce antibodies and were used to generate monoclonal ascites fluid. Seven Balb/C mice were primed by injection (18-gauge needle) with 200 µl pristane (Sigma Chemical Co.) ip, followed by a second injection 7 d later (31). After 3 d, 1 x 107 monoclonal hybridoma cells were injected ip. Four days later, ascite fluid was collected using an 18-gauge needle. Freezer stocks were made of the seven monoclonal hybridomas. A monoclone called 5F10 is described further here.
Indirect ELISA
Indirect ELISA was performed to determine the usefulness and relative dilution of each antibody. Relative dilution provides a starting dilution for other applications and is arbitrarily defined as the dilution before the highest dilution that significantly (P < 0.05) detects antigen above preimmune serum. Immulon-2 96-well microtiter plates were sensitized for 18 h at 4 C in duplicate with 500 ng rboISG15 antigen in a 50-µl volume. Antigen was diluted in carbonate coating buffer [35 mM NaHCO3 and 17 mM Na2 CO3 (pH 9.6)]. Plates were rinsed three times with PBS containing 0.05% Tween 20 (PBST) and blocked (60 µl) for 2 h at 37 C with blocking solution (1% BSA in PBS). Plates were rinsed three times with PBST and two times with distilled water. Preimmune serum (collected via the orbital plexus before immunization) or anti-rboISG15 monoclonal antibody was diluted in serum conjugate diluent [SCD; 75 mM NaCl, 0.5 mM EDTA, 50 mM Tris (pH 6.2), and 0.5% BSA] via doubling dilutions from 1:200 to 1:409,600. Antigen was reacted with diluted monoclonal antibodies or preimmune serum (50 µl) at 25 C for 1 h on a shaking platform. Unbound preimmune serum and antibody were decanted, and plates were rinsed three times with PBST and two times with distilled water. Mouse monoclonal antibodies were detected with horseradish peroxidase-conjugated goat antimouse polyvalent (IgG, IgM, IgA) immunoglobulin affinity-isolated antibody (1:3,000; Sigma Chemical Co.) at 25 C for 30 min on a shaking platform. Plates were again rinsed as described above, and 50 µl 3,3',5,5'-tetramethyl benzidine substrate [6 ml solution A (10 mM sodium acetate), 40 µl solution B (10 mM anhydrous citric acid), 63 µl 3',5,5'-tetramethyl benzidine (10 mg/ml in dimethyl sulfoxide; Sigma Chemical Co.), and 0.94 µl H2O2] were added for 5 min. The reaction was quenched with 50 µl 2 M H2SO4. Intensity of the reaction was measured at 450 nm. Duplicate plates were analyzed using paired t tests. Data were plotted as means ± SE to determine maximum dilution of antibody use.
Western blot analysis of monoclonal anti-ISG15 antibody
Western blotting (7, 9) was used to subjectively determine the relative titer for monoclonal antibodies. Relative dilution was considered the dilution before the highest dilution that detected native ISG15. Each Western blot contained rboISG15 (50 ng), rhISG15 (100 ng), bovine ubiquitin (100 ng), and lysates (20 µl) from bovine endometrial cells treated with 0 or 25 nM IFN. Monoclonal antibodies were diluted in Tris-buffered saline [TBS; 20 mM Tris (pH 7.5) and 150 mM NaCl] supplemented with 0.05% Tween 20 at 1:1000, 1:25,000, 1:50,000, 1:100,000, 1:200,000, and 1:400,000. Negative control Western blots were incubated with either no primary antibody or preimmune serum (1:1,000). Detection was accomplished using alkaline phosphatase-conjugated second antibodies (Promega Corp., Madison, WI) and nitroblue tetrazolium/ 5-bromo-4-chloro-3-indolyl-phosphate.
Ouchterlony double-immunodiffusion assay
Ascite fluid was tested for the presence of monoclonal antibodies and isotyped using the Ouchterlony double-immunodiffusion assay (32). Microscope slides were dipped in 1% Special Agar-Noble (Difco Laboratories, Detroit, MI). Agar was dissolved in single-strength Veronal buffer supplemented with 0.001% merthiolate and allowed to air dry in an up-right position. Three-millimeter sample wells were punched into the agar using a radial pattern. A central well was loaded with 10 µl crude ascite fluid, and surrounding wells were loaded with 7 µl of specific antiimmunoglobulin antibodies (Sigma Chemical Co.). Slides were placed in a humidified chamber, and antibodies were allowed to diffuse for 48 h. Soluble proteins were removed from the agar by incubating the slides in 0.85% saline for 24 h. Slides were placed at 37 C for 6 h to dry. Precipitated proteins were stained (6% Buffalo Black; Allied Chemical Co., New York, NY) for 5 min, fixed (four exchanges of 45% methanol and 10% acetic acid), and allowed to air dry for 1 h.
Immunohistochemical localization
Transverse sections of uterine horns were excised and fixed in 4% buffered paraformaldehyde for 24 h, followed by paraffin embedding and sectioning. Antigen retrieval was accomplished by microwaving two times for 5 min in 0.01 M citrate buffer (pH 6.0). ISG15 and its conjugates were localized using monoclonal 5F10 antibody (1:10,000) described here. Ubiquitin conjugates and the UBC E2-14K ubiquitin conjugating enzyme were localized using primary antibodies (1:2500) obtained from Dr. Art Haas (Medical College of Wisconsin, Milwaukee, WI). Primary antibodies were detected using Vectastain ABC kits (Vector Laboratories, Burlingame, CA). Slides were counterstained in hematoxylin, followed by lithium carbonate (1%), and coverslipped in Crystal Mount (Fisher Chemical, Denver, CO). Positive staining appeared red.
Western blot analysis of endometrial lysates
Tissue lysates were prepared by homogenizing 100 mg endometrium in 1 ml Laemmli buffer (33). Lysates (20 µl/lane) were loaded onto one-dimensional PAGE gels, transferred to 0.2 µm nitrocellulose in single-strength Towbin buffer, and Western blot detection was performed using monoclonal anti-ISG15 (5F10) at 1:200,000 and anticonjugated ubiquitin at 1:10,000. Second antibodies against mouse or rabbit were conjugated to alkaline phosphatase and used at a 1:10,000 dilution (Promega Corp.). Immunoreacting bands were visualized using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate solution (Promega Corp.). Western blots were scanned and quantitated using UNSCANIT (Silk Scientific, Salt Lake City, UT).
Electron microscopy and immunogold staining
Endometrium was cut into 1-mm pieces and fixed overnight in 1% glutaraldehyde/0.1 M sodium phosphate (pH 7.4) and then washed with several changes of sodium phosphate buffer before being dehydrated (graded series of ethanol), cleared (propylene oxide), and embedded in EMBED 812 resin (EMS, Fort Washington, PA). Polymerization of resin occurred at 35 C for 8 h, 45 C for 12 h, and 60 C for 8 h. Thin sections were collected on 300-mesh nickel grids coated with 0.5% pioliform in ethylene dichloride. The grids were then etched in 10% H2O2/20 mM Tris (pH 7.4) and washed in 0.1% Triton X-100. Grids were blocked in 1% BSA + 5% goat serum in TBS for 1 h at room temperature. Grids were drained and incubated in monoclonal anti-ISG15 antibody (5F10; 1:200) in 1% goat serum/TBS/BSA buffer. They were washed several times in TBS/BSA buffer and then incubated in goat antimouse (conjugated to 12-nm gold particles) secondary antibody for 1 h at 37 C. The grids were rinsed in TBS/BSA buffer and 0.1 M sodium phosphate (pH 7.4) and postfixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer for 10 min at room temperature. Finally, the grids were washed in HPLC-grade water and poststained with 1% uranyl acetate (10 min) and lead citrate (15 sec). Tissue sections were viewed on a Philips transmission electron microscopy (TEM) (magnification up to x16,900; 60 kV; exposure, 2.5 sec).
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Results |
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Localization of ISG15, ubiquitin, and ubiquitin E2-14K enzyme in bovine endometrial tissue
ISG15 was localized in endometrium from d 18 pregnant and nonpregnant cows (Fig. 1
). In endometrium from d 18 nonpregnant cows, only subtle staining for ISG15 was detected in the lumenal and glandular epithelia. This is in contrast to the dramatic increase in the amount of ISG15 staining found in the endometrium from d 18 pregnant cows. Intense staining for ISG15 was present in the glandular epithelium and the sublumenal stromal cells, with less staining occurring in the lumenal epithelium.
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A more expanded analysis of staining of ISG15 in uterine cross-sections during pregnancy and the estrous cycle is shown in Fig. 2. On d 18–23 of pregnancy, ISG15 and its conjugates were localized most heavily in the stratum compactum surrounding the lumenal epithelium and the glandular epithelium throughout the stratum spongiosum. Interestingly, by d 45–50, ISG15 and its conjugates were still detected in the stratum compactum immediately beneath the lumenal epithelium with no staining in the glandular epithelium. Note that this is a period that is long after trophoblast-derived IFN is secreted. Staining for conjugated ubiquitin was consistently high in the lumenal epithelium and glandular epithelium when compared with stromal cells but did not change significantly over the days of the estrous cycle or pregnancy that were examined in this experiment.
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). Very few morphological differences were noted in these cells across pregnancy status. Similar rough endoplasmic reticulum and mitochondria were found throughout these cells. There was no aggregation of either organelle around the nucleus. Rather, they were spread sparsely throughout the cells. There was a large amount of vacuole-like structures that were usually found surrounding the nucleus in all glandular cells. Lumenal epithelial cells also retained similar morphological features regardless of pregnancy status. The only minor difference was the presence of vacuole-like structures that contained an osmophilic substance in uterine cross-sections from pregnant cows. The morphology of stromal cells also did not appear to change due to pregnancy status on d 18.
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). Positive staining was observed as small, uniform black dots and also was noted, to a lesser degree, in uterine cross-sections from d 18 nonpregnant cows. The negative controls for this experiment received no primary antibody. These cells did have some limited staining that was located throughout the cell and in the free resin that was indicative of background staining.
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). Free (17 kDa) and conjugated (>30 kDa) ISG15 were detected using 5F10 monoclonal antibody and Western blot. Both free and conjugated ISG15 were higher (P < 0.05) on d 17 of pregnancy when compared with the estrous cycle and remained elevated from d 18–23. Free ISG15 declined sharply from d 23–45 to levels that were much lower but still higher than background levels noted in nonpregnant cows. Conjugated ISG15 followed the pattern of expression for free ISG15 but did not differ when comparing endometrial extracts from nonpregnant cows to d 45 of pregnancy. Neither free nor conjugated ISG15 differed on d 50 of pregnancy when compared with levels observed during the estrous cycle. Conjugated ubiquitin was also quantified and compared in pregnant and nonpregnant cows and was not affected by pregnancy in the cow.
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Discussion |
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. Described here, for the first time, is the intracellular expression of ISG15 and its conjugates in bovine endometrium from d 17–50 of pregnancy. Intracellular ISG15 protein followed a similar pattern of expression as ISG15 when released from cultured explants representing d 15–26 of pregnancy (7). However, on d 45 of pregnancy, intracellular ISG15 was difficult to detect when compared with elevated intracellular levels of proteins that become conjugated to ISG15. Because conjugated ISG15 remains in the endometrium as late as d 45 in the face of declining or negligible free ISG15, it is postulated that one function of ISG15 is to stabilize proteins rather than to target them to degradation as described for polyubiquitination.
One goal of the present experiments was to localize ISG15 to cells in the endometrium during the estrous cycle and pregnancy. In situ hybridization of ISG15 mRNA in both bovine (10) and ovine (12) uterine cross-sections revealed that ISG15 mRNA was localized to deep and shallow glandular and stromal cells as well as to the myometrium during time of maximal release of IFN from bovine (d 18) and ovine (d 16) conceptuses. Based on these results, we hypothesized that immunohistochemical localization of ISG15 to uterine tissues would be similar. In the present experiments, development and use of a monoclonal antibody against recombinant bovine ISG15 in immunohistochemistry of bovine uterine cross-sections revealed the following. Light staining for ISG15 was found in the lumenal epithelium. More heavily stained cells were found in both shallow and deep endometrial glands with a more diffuse staining pattern in corresponding stromal cells. Staining for ISG15 also was found in the myometrium. Use of the polyclonal antibovine ISG15 antibody revealed similar results with slightly higher background staining (data not shown). The lack of intense staining for ISG15 in the lumenal epithelium when compared with more intense staining in the glandular epithelium is consistent with reports by others when evaluating IFN-induced genes such as Mx and 2',5'-oligoadenylate synthase (12, 13, 14, 35, 36, 37, 38, 39).
One explanation for the more intense deep glandular staining for ISG15 might be that transcription factors suppressing interferon-stimulated genes, like interferon regulatory factor-2, are active in the lumenal epithelium (35, 36). Another explanation might be that the lumenal epithelium is exposed to such high levels of IFN that the receptor and associated signal transduction pathways become uncoupled. For example, the conceptus releases about 0.25 mg IFN into the uterine lumen on d 18 of pregnancy in the cow (37). This level of IFN is in the micromolar range. The dissociation constant of the type 1 interferon receptor is in the nanomolar range. Exposure of receptors to 10–100 times the dissociation constant can result in down-regulation of the receptor and desensitization or uncoupling of signal transduction.
Presence of a signal for ISG15 in the myometrium in uterine cross-sections also is very interesting. For a signal to be present in this tissue of the uterus, which is several cell layers beneath the lumenal epithelium, one of two responses might be occurring. First, fenestrations exist in the basal lamina of the lumenal epithelium (38). So, IFN could diffuse through these sites and reach the glandular and stromal cells and, depending on concentration, could also reach the myometrium. An alternate pathway might be through cell-to-cell communication in which IFN stimulates one cell to release a signal to another cell, which then essentially cascades deeply into the myometrium in which the ISG15 gene is activated.
Use of antibodies against conjugated ubiquitin (39) revealed that ubiquitin conjugates were present in uterine cross-sections but did not change in amount when comparing uterine cross-sections from pregnant with nonpregnant cows. The ubiquitin conjugates were selected for this experiment as controls to determine whether the related ubiquitin conjugating system was regulated by pregnancy. The antibody against ubiquitin when conjugated to proteins is very specific and does not detect free ubiquitin or ubiquitin-like proteins. This should be contrasted with several commercially available antibodies that recognize ubiquitin, ubiquitin when conjugated to proteins, and other ubiquitin homologs (i.e. ISG15, Nedd 8, and Sumo-1). The cross-reactivity of antiubiquitin antibodies with other ubiquitin homologs should be considered when interpreting changes in protein expression because an increase or decrease might simply reflect a change in one of the immunoreacting homologs.
Implantation in humans is intrusive with the embryo invading the endometrial epithelium at d 7. Cows, in contrast, exhibit a superficial implantation in which the embryo attaches to the uterus in several sites via the placenta. In primates, stromal cells enlarge and proliferate, creating a decidual cell response similar to that of mice. Regardless of the mode of implantation, it is evident that up-regulation of ISG15 is a universal response during pregnancy in mammals, particularly in light of recent reports of ISG15 in endometrium from pregnant mice (17) and pigs (16) in addition to primates (5) and ruminants.
In addition to having a role during early pregnancy in mammals, ISG15 also has been found to be immunolocalized to the cytoplasm of lymphoid cells, neurons from brain stem, neuromuscular junctions, stomach parietal cells, collecting ducts of the kidney, epithelium of the thyroid, faint granular staining in visceral smooth muscle, and striations of skeletal muscle and cardiac muscle (40). Because ISG15 has been localized, to some degree, in many diverse yet critical tissues, it could be assumed that it plays a major role in cell physiology/biology. In normal tissues, ISG15 was stained in a cytoplasmic punctate pattern. Loeb and Haas (41) reported that ISG15 was associated with intermediate filaments. Perhaps ISG15 is acting as an intermediate filament-associated protein.
The morphology of bovine endometrial epithelial cells was compared in d 18 pregnant and nonpregnant cows using TEM. ISG15 is expressed in these epithelial cells with maximum expression around d 18. Although fewer cells were examined in the present experiment, the morphology results obtained closely resemble those found by others. King et al. (42) examined endometrial epithelial cells from nonpregnant cows and pregnant cows from d 17–45. The nonpregnant cells were pseudostratified columnar cells and their appearance did not differ from d 17 cells. The only variation of d 18 pregnant endometrial cells from the nonpregnant cells was that they became lower columnar cells. Wathes and Wooding (43) found that epithelial cells on d 18 of pregnancy had an average height of 20–25 µm. Ultrastructure of these cells was similar to cells of nonpregnant cows. Approximately 3% of epithelial cells from d 18 pregnant cows contained two nuclei. This observation was not noted in the current study, but odd-shaped and occasional bilobed nuclei were observed (data not shown). Examination of cellular localization of ISG15 using TEM and immunogold labeling revealed a diffuse staining pattern in uterine glandular epithelial cells. Rather than being discretely localized to a cellular compartment or organelle, specific staining for ISG15 was found throughout these cells from uterine cross-sections on d 18 of pregnancy. For example, specific immunogold staining of ISG15 was observed in the plasma membrane, cytoplasm, mitochondria, endoplasmic reticulum, perinuclear areas, and the nucleus.
It is concluded from this study that ISG15 has diverse function in the endometrium, which is not limited in scope to a specific cellular compartment or organelle. This diverse localization and cellular function for ISG15 in the endometrium is consistent with recent reports that disruption of the ISG15 conjugation pathway leads to development of hydrocephalus a few weeks after birth in mice (44). Dysregulation of ISG15ylation in mice leads to decreased life expectancy, brain cell injury, and hypersensitivity to interferon (44). This is due, in part, to the role of ISG15 in regulating signal transduction events (45, 46). Described herein is the expression of endometrial ISG15 during early pregnancy and the initial stages of implantation. Because ISG15 was specifically localized to many compartments of glandular or stromal cells, it is concluded that the function of ISG15 is diverse rather than being restricted to a cellular organelle (e.g. mitochondria) or compartment (e.g. perinuclear). This would be consistent with early reports that ISG15ylation was involved with the cytoskeleton (41) and more recent reports that it is involved with diverse signal transduction pathways (45, 46). In either case, it is clear from the present studies that a general endometrial response to pregnancy is massive endometrial expression of ISG15. Because the expression of ISG15 and its conjugates continues long after release of IFN from the bovine conceptus has decreased, it is hypothesized that this response prepares the uterus for the implanting embryo. This hypothesis will be tested using murine null (44) and bovine models in future experiments.
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Acknowledgments |
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Footnotes |
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Present address for A.L.C.: UniPath, Denver, Colorado 80222.
Present address for J.K.P.: Vincent Center for Reproductive Biology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114.
Abbreviations: FBS, Fetal bovine serum; HAT, hypoxanthine aminopterin thymidine; HBSS, Hank’s balanced salt solution; IFN, interferon-; ISG, interferon-stimulated gene; PBST,PBS containing Tween 20; TBS, Tris-buffered saline; TEM, transmission electron microscopy.
Received August 20, 2003.
Accepted for publication October 8, 2003.
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References |
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in the uterus during early pregnancy. J Reprod Fertil Suppl 54:329–339[Medline]
inducible ubiquitin cross-reactive protein in the ovine uterus. Biol Reprod 61:312–318 and progesterone regulate ubiquitin cross-reactive protein expression in the ovine uterus. Biol Reprod 62:622–627This article has been cited by other articles:
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