Molecular Human Reproduction, Vol. 5, No. 10, 966-972,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular aspects of pregnancy |
Ubiquitin cross-reactive protein gene expression is increased in decidualized endometrial stromal cells at the initiation of pregnancy
1 Department of Obstetrics & Gynaecology, School of Human Development and 2 School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK and 3 Department of Obstetrics & Gynaecology, University of Sydney, Westmead Hospital, Sydney, NSW 2145, Australia
Abstract
Ubiquitin cross-reactive protein (UCRP; also known as ISG15) is an interferon-upregulated protein implicated in the response to viral infection. An equivalent protein has been demonstrated within the bovine uterus in early pregnancy in response to conceptus-derived interferon-
. We have previously shown the upregulation of UCRP within decidualized stromal cells of the human and non-human primate endometria. We now show that an increase in UCRP gene expression within the decidualized stromal cell accompanies these increased protein concentrations. Using Northern blotting techniques we demonstrate basal concentrations of URCP mRNA within the non-pregnant endometrium and an increase in signal within some decidual specimens of first trimester decidua. This signal may represent increased URCP transcription within the sub-population of stromal cells that undergo decidualization, since this cell type exhibits an increase in UCRP message as detected by in-situ hybridization.
endometrium/ISG15 (UCRP)/human/pregnancy/ubiquitin cross-reactive protein
Introduction
Ubiquitin cross-reactive protein (UCRP, also known as ISG15) is an interferon (IFN)-upregulated, 15 kDa protein that is structurally similar to a head to tail repeat of ubiquitin (Ub). Covalent attachment of Ub to intracellular proteins targets them for degradation by the 26S proteasome (Hershko and Ciechanover, 1998
). UCRP retains the C-terminal RGG sequence of Ub that is necessary for activation and conjugation of Ub and Ub-like proteins to target proteins (Haas et al., 1987). Indeed, UCRP is known to conjugate to intracellular proteins (Loeb and Haas, 1992) although the role of this protein modification is not yet known. UCRP can be released from cells and appears to be immunoregulatory (D'Cunha et al., 1996) and may be a factor in the induction of the antiviral response of human cells to type 1 IFN (Korant et al., 1984). IFN-
has less efficacy than IFN-
or -ß in the induction of UCRP production in human fibroblast cells (Taylor et al., 1996), but may in turn be synthesized by human peripheral blood cells in response to extracellular UCRP (Recht et al., 1991).
The major cell types in the human endometrium are stromal and epithelial cells, with both glandular and luminal epithelial cell types present. In the first trimester of pregnancy, the endometrium undergoes modification to form the decidua, a tissue containing several populations of cells. Stromal cells similar to those in the non-pregnant endometrium are present along with cells that have undergone transformation into the true decidual cell type, with enlarged cytoplasm, secretory properties and a basal membrane surrounding each cell (Kearns and Lala, 1983). Endometrial glands remain in those regions of tissue rich in stromal cells (the decidua spongiosa), while within the decidua compacta (regions of true decidual cells) glands degenerate until few remain in the second half of gestation. Spiral arteries comprising endothelial cells, and bone marrow-derived cells (which infiltrate into the decidua during early pregnancy) are also present within this tissue.
A molecule homologous to human UCRP (hUCRP) has been detected in tissues of the bovine uterus, and named bUCRP (Austin et al., 1996, Perry et al., 1999). bUCRP is upregulated in response to IFN-, a molecule produced by the developing conceptus and necessary for the maintenance of pregnancy in cows. Upregulation of bUCRP is observed at the level of transcription (Hansen et al., 1997) and in increased concentrations of both conjugated and monomeric protein (Naivar et al., 1995; Johnson et al., 1998). bUCRP is secreted by uterine tissues in vitro in response to IFN-, and may be detected in uterine flushings from pregnant animals (Austin et al., 1996).
We have recently demonstrated the upregulation of hUCRP-conjugated proteins in the human and non-human primate endometrium during decidualization at the initiation of pregnancy (Bebington et al., 1999). In the absence of IFN, UCRP mRNA and protein have been demonstrated at low concentrations in vitro in cell lines (MG-63, A549 and Hep-2) (Ahrens et al., 1990; Loeb and Haas, 1992), but both are seen to accumulate in response to IFNs (Farrell et al., 1979). This study was designed to investigate whether, in response to decidualization, increased UCRP gene expression occurs, in addition to the increased concentrations of protein observed previously (Bebington et al., 1999). Due to the heterogeneous nature of human endometrium and the decidual cell-specific localization of UCRP protein (Bebington et al., 1999) the technique of in-situ hybridization was employed in order to localize the site of UCRP production as well as Northern blot analysis to determine total tissue concentrations of UCRP mRNA.
Materials and methods
Probe preparation and labelling
Probe for UCRP mRNA was a generous gift of Prof. Haas (University of Wisconsin, Wisconsin, USA), and was provided in the form of a modified pGEM-T plasmid vector (Promega, Southampton, Hampshire, UK) with probe to the hUCRP sequence inserted between BamHI and HindIII restriction sites. The sequence of the insert was checked by automated DNA sequencing (Applied Biosystems 373A DNA Sequencer, Foster City, CA, USA) in both directions using pUC/M13 primers. Sequencing was carried out by the Biopolymer Synthesis and Analysis Unit of the School of Biomedical Sciences, Nottingham University. Approximately 90% of the sequence of the insert was reliably determined and found to be identical to the complementary published sequence of hUCRP.
Probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as a control for loading and transfer of mRNA, was amplified by the polymerase chain reaction (PCR) with sequence-specific primers according to the published sequence (Stratagene, Amsterdam, The Netherlands), and was labelled in a manner similar to that described for UCRP probe.
Probe labelling for in-situ hybridization was performed using the random prime method (Feinberg and Vogelstein, 1983) incorporating fluorescein. Components for DNA labelling were obtained from the DNA colour kit for non-radioactive in-situ hybridization (Amersham Ltd, Chalfont, Bucks, UK), and labelling was performed according to the supplier's protocol provided. Northern blot protocols employed probe labelled with [32P]dCTP (ICN Biomedicals Ltd, Oxfordshire, UK) by random prime methods using a DNA labelling kit (Pharmacia Biotech Ltd, Bucks, UK) with unincorporated radioactivity removed using a Sephadex spin column (NickSpin Column, Pharmacia Biotech).
Sample collection
Non-pregnant endometrial specimens were collected from pre-menopausal women undergoing hysterectomy at the Queen's Medical Centre for indications other than those of malignancy (n = 5, embedded for in-situ hybridization protocols; n = 5, frozen for Northern blotting; n = 4 frozen for Western blotting). Endometrial tissue was carefully removed from the underlying myometrium immediately after removal. The stage of the menstrual cycle of each tissue was estimated by the time of the last menstrual period and was confirmed by histological dating performed by experienced clinical pathologists. First trimester decidua was obtained by dissection from the products of conception following therapeutic terminations of pregnancy at the Queen's Medical Centre (n = 5, processed for in-situ hybridization; n = 8, frozen for Northern blotting; n = 5 frozen for Western blotting, gestation 7–12 weeks). The duration of pregnancy was calculated from the date of the last menstrual period. Informed consent was obtained from all subjects and all work was approved by the Ethics Committee of Queen's Medical Centre, Nottingham.
In-situ hybridization
Tissues were fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; 10 mmol/l phosphate buffer, 2.7 mmol/l KCl, 137 mmol/l NaCl, pH 7.4) within 30 min of removal. After fixation for 24–48 h, tissues were dehydrated, embedded in paraffin and 4 µm sections cut and mounted on glass microscope slides. Slides were pre-treated with 3-aminopropyl triethoxysilane (Sigma Chemical Co., Poole, UK) in order to prevent tissue loss during the in-situ hybridization protocol.
Sections were de-waxed in xylene and rehydrated through a series of ethanols before rinsing in PBS, 10 min immersion in 0.2 mol/l HCl and rinsing in 0.01% (v/v) Triton X-100 (Sigma) in PBS. Slides were incubated in 4% (w/v) paraformaldehyde in PBS for 5 min at room temperature. Proteinase K (Boehringer Mannheim Ltd, East Sussex, UK) at a concentration of 5 µg/ml was applied to sections for 5 min at 37°C to weaken cross-links produced by the fixation processes. Sections were washed in 0.2% (w/v) glycine in PBS for 5 min and rinsed in PBS prior to hybridization. Repeated washes in PBS were performed between each stage of pre-treatment.
Hybridization was performed overnight at 42°C in a humidified chamber. Hybridization buffer contained Denhart's solution [0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrrolidone, 0.02% (w/v) bovine serum albumin (BSA, Sigma)], 300 µg/ml salmon testis DNA and a novel rate-enhancing compound buffered with saline sodium citrate (SSC) that was a component of the DNA colour kit for in-situ hybridization (Amersham). Wash solution consisted of SSC (0.15 mol/l sodium chloride, 0.015 mol/l sodium citrate, pH 7.0). Washes were performed twice in SSC containing 0.1% (w/v) sodium dodecyl sulphate (SDS) for 5 min at room temperature with agitation and twice in 0.2xSSC containing 0.1% (w/v) SDS at 42°C for 10 min, followed by a 5 min wash in Tris-buffered saline (TBS; 50 mmol/l Tris–HCl, 150 mmol/l NaCl, pH 7.5) prior to immunodetection of bound probe.
Non-specific antibody binding was prevented by incubation of slides in blocking solution [1% (w/v) BSA, 0.1% (v/v) Triton X-100 in PBS] for 30 min. All incubations were performed at room temperature in a humidified chamber followed by repeated washes in TBS unless stated otherwise. Primary antibody (mouse antifluorescein; Dako Ltd, High Wycombe, UK) was used at 1:75 in blocking solution for 1 h. The secondary antibody employed was biotinylated rabbit anti-mouse IgG (Dako) used at a dilution of 1:500 in TBS for 30 min, followed by streptavidin conjugated to alkaline phosphatase (Dako) at 1:100 in TBS for 30 min. Tissues were washed thoroughly in TBS and then immersed in detection buffer (100 mmol/l Tris–HCl, 100 mmol/l NaCl, 50 mmol/l MgCl2, pH 9.5) prior to colour development with nitroblue tetrazolium (2 mg/ml) and bromochloroindolyl phosphate (0.1 mg/ml) in the presence of an inhibitor of endogenous alkaline phosphatases (1 mmol/l levamisole; Sigma). Slides were mounted using an aqueous mountant (UV-Inert; Hopkin and Williams, Essex, UK), and staining was observed using bright-field microscopy and photographed with Kodak 200 film.
Four negative controls were performed: (i) omission of probe (incubation of slides in the presence of hybridization solution alone), (ii) omission of primary antibody, (iii) antibody detection without hybridization protocols and (iv) pre-treatment of tissue with RNase A type I just prior to hybridization. This control involved the incubation of slides for 30 min at 37°C with RNase A type I [0.01% (w/v) in SSC, Sigma] followed by two washes in SSC and one wash in PBS, each of 5 min.
Northern blots
Freshly collected tissues were immediately frozen in liquid nitrogen and were stored at –80°C until use. Total RNA was extracted from tissues using a commercially available kit (RNA isolation kit; Fluka, Dorset, UK) adapting standard protocols adapted from Chomczynski and Sacchi (1987). RNA blotting was performed according to a previously published glyoxal and Acridine Orange method (McMaster and Carmichael, 1977). Briefly, 20 µg total RNA (as estimated by spectrometric analysis of samples) from each tissue was heated in the presence of dimethylsulphoxide and glyoxal in phosphate buffer (10 mmol/l, pH 7.0) and was electrophoresed on an agarose gel [1% (w/v) agarose, 10 mmol/l iodoacetic acid, in 10 mmol/l phosphate buffer, pH 7.0] at 40 V for 2–3 h with circulation of electrode buffers. RNA was transferred by capillary action to a nylon membrane (Hybond-N; Amersham), the membrane was baked at 80°C for 2 h, and the membrane was exposed to UV light at 245 nm (Stratagene).
Pre-hybridization was performed at 60°C for 1 h in commercially available hybridization solution (QuikHyb; Stratagene) followed by overnight hybridization at 60°C. Hybridization solution consisted of QuikHyb containing salmon testis DNA (Sigma) and radio-labelled probe. Repeated stringency washes in 2xSSC, 0.1% (w/v) SDS (with agitation at room temperature) and in 0.1xSSC, 0.1% (w/v) SDS (with agitation at 60°C) removed unbound probe before the membrane was exposed to photographic film (Amersham) at –80°C for 2–3 days. Densitometry was performed using Aida software on a Fugi-Raytek densitometer.
The size of mRNA species detected was calculated by concurrent electrophoresis of RNA size markers (Gibco, Paisley, UK), detached from the body of the gel prior to transfer, labelled using ethidium bromide (Sigma) at 0.5 µg/ml in 0.1 mol/l ammonium acetate, and visualized using UV light (312 nm). RNA loading was normalized by comparison with matched lanes of the blot, hybridized with probe for GAPDH using an identical protocol. The optical density of bands generated when transfers were exposed to UCRP or GAPDH probe were compared and a ratio obtained.
Western blots
The primary antibody employed was a polyclonal antibody raised in sheep against a synthetic fragment of bUCRP, LVRNDKGRSSPYEVQLKQ (Johnson et al., 1998), and was a generous gift of Dr T.Hansen (University of Wyoming, Wyoming, USA).
Frozen tissue was rapidly homogenized in electrophoresis sample buffer (62.5 mmol/l Tris–HCl, pH 6.8, 2.3% (w/v) SDS, 10% (v/v) glycerol, and 0.01% (w/v) bromophenol blue containing protease inhibitors (3 mmol/l pepstatin, 4 mmol/l EDTA, 2 mmol/l PMSF, 5 mmol/l N-ethylmaleimide). 5% (v/v) ß-mercaptoethanol was then added to each sample and the tubes boiled for 5 min before storage at –20°C prior to use. Relative protein concentration of the samples dissolved in electrophoresis sample buffer was obtained by precipitation with tricarboxylic acid followed by measurement of turbidity at 550 nm (Karlsson et al., 1994). Equal amounts of protein were loaded into each well of a 10 lane, 12.5% (w/v) acrylamide gel (acrylamide: bis-acrylamide, 37.5:1) surmounted by a 4% (w/v) stacking gel (Laemmli, 1970). Estimation of molecular weight was possible due to the concurrent electrophoresis of coloured molecular weight markers (Amersham). Electrophoresis was carried out at 25 mA for ~1.5 h followed by overnight electro-transfer of the proteins to nitrocellulose (Hybond C-Super; Amersham) in 25 mmol/l Tris, 192 mmol/l glycine, 20% (v/v) methanol and 0.1% (w/v) SDS.
Following transfer, nitrocellulose membranes were incubated for at least 1 h with 5% (w/v) milk powder and 0.1% (v/v) Tween 20 (Sigma) in TBS (TBS/Tween) at room temperature to prevent non-specific binding of antibodies. Immunostaining was carried out in blocking buffer containing the previously described polyclonal sheep serum containing antibodies raised against UCRP (diluted 1:5000 from the stock solution provided) for 2 h at room temperature. The antiserum was removed and the transfer was washed repeatedly in TBS/Tween before addition of the peroxidase-conjugated protein-G (BioRad, Hemel Hempstead, UK) diluted 1:5000 in TBS/Tween and incubation for 1 h. After further washes in TBS/Tween, a final rinse was carried out in TBS alone followed by detection of bound peroxidase-conjugated secondary antibodies using an enhanced chemiluminescent system (NEN-Du Pont, Stevenage, UK). Hyperfilm-ECL (Amersham) was exposed to the transfer and developed to produce a permanent record of the results.
Results
Northern blots performed on tissue homogenates of non-pregnant and pregnant endometrium demonstrate that an RNA species of the predicted size for UCRP is detected in all samples (Figure 1A). In addition to the 0.7 kb band, a much fainter band of ~5.6 kb is also observed upon longer exposure (data not shown). RNA of 1.3 kb is detected when hybridized to a probe to the housekeeping gene, GAPDH (Figure 1B). Omission of probe eliminates the signal (data not shown). Following densitometry, the optical density of bands corresponding in size to UCRP transcript was compared with the optical density of the band corresponding to GAPDH transcript for the same sample (Table I).
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Western blotting for UCRP-containing polypeptides demonstrates a species immunoreactive with anti-UCRP at ~46 kDa which is present in all decidual samples and to a lesser extent in two of four non-pregnant endometrial samples (Figure 2
). Additional higher molecular weight species are present uniquely in pregnant tissues. Omission of primary antibody eliminates the signal (data not shown).
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Immunoreactivity with anti-fluorescein following in-situ hybridization with fluorescein-labelled probe for UCRP mRNA is seen within the cytoplasm of decidual cells from first trimester pregnancy decidua (Figure 3A and C
) but is not seen within non-decidualized stromal cells of the pregnant decidua (Figure 3B) nor within non-pregnant endometrium (Figure 4A and B). Neither endometrial glandular epithelium nor stromal cells demonstrate reactivity to the probe (Figure 4A). Signal is not observed in negative controls (Figure 3D, E, F and G), and omission of antibody or of hybridization protocols eliminates signal (Figure 3B). Myometrial tissues within the same section as non-pregnant endometrial specimens exhibit moderate levels of hybridization (Figure 4C). No difference in levels of hybridization is seen within endometria from different stages of the menstrual cycle (data not shown), but considerable variation in mRNA concentrations within different samples of decidua is observed. Non-decidualized stromal cells of the decidua spongiosa are not reactive with the probe (Figure 3B). Glandular tissues within the decidua demonstrate moderate reactivity with the probe, but may contain low concentrations of endogenous alkaline peroxidase since occasional reactivity is also seen in negative control tissues (data not shown).
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Discussion
Increased concentrations of UCRP mRNA in pregnant endometrial samples were not easily demonstrated when analysed by Northern blotting (Figure 1
). Concentrations of UCRP mRNA (normalized with respect to GAPDH mRNA concentrations) were slightly increased in pregnancy (Table I), with mean optical density values of 1.70 (non-pregnant, SD = 0.52, n = 3) and 1.82 (pregnant, SD = 0.27, n = 5), but these differences are not statistically significant (P > 0.5; Student's t-test). There is considerable variation in normalized tissue content of UCRP mRNA within non-pregnant (range 1.4–2.3) and within the pregnant groups (range 1.4–2.1). A single sample collected at 9 weeks of pregnancy (Figure 1A, lane 5) apparently contains very low concentrations of UCRP mRNA (1.4), whereas a single non-pregnant sample (Figure 1A, lane 1) apparently contains high concentrations of UCRP mRNA (2.3). This phenomenon may partly be explained by the heterogeneity of the tissue examined. Decidualized stromal cells were the only cell type observed to contain UCRP mRNA upon in-situ hybridization (Figure 3A and C), and the proportion of these cells (in relation to non-decidualized stromal cells or glandular tissues) within biopsies of tissue collected may vary considerably. An indication of the proportion of decidualized stromal cells in a given tissue sample could be provided by probing with a suitable marker of decidualization. However, there are potential difficulties with obtaining such a marker. The intermediate filament protein vimentin may be present in decidualized stromal cells but is also apparently present in non-decidualized stromal cells (Oliver et al., 1999), while a major secretory product of the decidualized stromal cell, IGFBP-1, shows varied abundance within this heterogeneous population of cells (Waites et al., 1989). The use of purified stromal cells from non-pregnant or pregnant endometria for Northern blotting would clarify the source of signal detected. Similarly the use of stromal cells decidualized in vitro would allow more precise study of the potential effects of decidualization on the expression of UCRP mRNA. These investigations are currently underway in our laboratory. However, we were able to demonstrate by Northern blotting that the UCRP probe hybridized to a transcript of the appropriate size for UCRP mRNA in all tissue samples examined (Figure 1A).
Following electrophoresis and immunoblotting, anti-UCRP immunoreactive polypeptides can be detected in pregnant and non-pregnant endometrium (Figure 2). The most prominent anti-UCRP immunoreactive polypeptide has a mass of ~46 kDa and probably consists of UCRP conjugated to an unknown cellular protein (Figure 2). We have detected a UCRP-containing polypeptide of this size in endometrium and decidua previously with an antibody raised against hUCRP (Bebington et al., 1999). The concentrations of the 46 kDa UCRP conjugate apparently increase during pregnancy, although it is also clearly present in non-pregnant secretory phase endometrium (Figure 2, lanes 3 and 4). In addition, some higher molecular weight species are present in decidua but not non-pregnant endometrium (Figure 2, lanes 5 and 7). These suggest that increased concentrations of conjugated UCRP protein accompany decidualization. The sheep primary antibody employed in this study was raised against a synthetic peptide corresponding to a sequence present in bUCRP, LVRNDKGRSSPYEVQLKQ (Johnson et al., 1998). The corresponding sequence in hUCRP (LVRNNKGRSSTYEVRLTQ) is different in only four residues. In addition, the major polypeptide detected by the sheep antibody to the bUCRP peptide is the same size as the major polypeptide we detected (Bebington et al., 1999) using an affinity-purified rabbit polyclonal antibody to recombinant hUCRP (Loeb and Haas, 1994), which suggests that the antibody used in this study detects hUCRP. Although increased UCRP protein concentrations may be seen in pregnant endometrial samples, immunocytochemistry demonstrates the decidual cell-specific expression of UCRP more clearly than immunoblotting (Bebington et al., 1999).
As samples collected for Northern and Western blotting are heterogeneous, containing both decidual and stromal cells, we employed in-situ hybridization to look for cell-specific expression of UCRP at the mRNA level. Hybridization of a probe for UCRP mRNA to tissue sections is increased in decidual cells from first trimester endometrium compared to non-pregnant stroma (Figure 3A and C versus Figure 4A and B). No immunoreactivity with anti-fluorescein is seen in the absence of probe or if tissue RNA is pre-digested with ribonuclease A (Figure 3D, E, F and G) suggesting that the immunoreactivity observed is indeed due to the presence of UCRP mRNA hybridizing with the probe. We cannot exclude the possibility that UCRP mRNA is present in non-pregnant stroma at concentrations below the limits of detection of our technique. The lack of reactivity within human endometrium is not due to mRNA degradation within these specimens, as adjacent myometrial tissues demonstrate detectable, though variable, concentrations of mRNA (Figure 4C). The fact that mRNA concentrations appear to vary between decidua from different patients, and indeed between adjacent regions of decidual cells in the same section of tissue, is not surprising since variability in protein and mRNA concentrations in decidual cells has been observed in previous studies and is characteristic of this tissue (Waites et al., 1989). These variations, coupled with variable ratios of decidual and stromal cells, and possibly myometrial contamination, probably accounts for the variable concentrations of UCRP mRNA and protein seen in pregnant and non-pregnant tissue (Figures 1 and 2). We did not observe any clear gestational age-related changes in UCRP expression. We were unfortunately unable to obtain tissue prior to 7 weeks of pregnancy which would have provided a greater insight into the very early stages of implantation.
The absence of reactivity within human endometrium to in-situ hybridization under these conditions may not indicate complete absence of this species; indeed results from Northern blotting (Figure 1A, lanes 1, 2 and 3) indicate that UCRP mRNA is present in non-pregnant tissue although this, at least in part, could be due to myometrial contamination. Other studies have shown that UCRP mRNA is present constitutively in the absence of stimulatory signals (Ahrens et al., 1990; Loeb and Haas, 1992). Indeed, we have also demonstrated the presence of low concentrations of a 46 kDa protein species containing UCRP within homogenates of non-pregnant human endometrium (Figure 2, lanes 3 and 4).
The primary source of IFN- within the endometrium is T-lymphocytes, located in the lymphoid aggregates in the stratum basalis, the glandular epithelium and throughout the stroma of basal and functional layers (Stewart et al., 1992). A recent study has demonstrated that endometrial polymorphonuclear leukocytes also produce IFN- (Yeaman et al., 1998). Some studies have indicated that uterine natural killer cells and macrophages may also produce this cytokine (Robinson et al., 1985; Pestka et al., 1987) but within the normal endometrium these cells are not immunoreactive for IFN- (Stewart et al., 1992). Endometrial IFN- may be involved in a complex network of endometrial cytokines (Lim et al., 1998; Nasu et al., 1998). Although IFN- does not induce UCRP expression as strongly as IFN- or -ß, it does exert a minimal effect (Haas et al., 1987) and may explain the low but detectable concentrations of RNA and protein observed in non-pregnant endometria. It is possible that the administration of prostaglandin pessaries prior to termination of pregnancy has affected our findings. Exogenous prostaglandins may be involved with the complex network of uterine cytokines; however, no work has yet been published on any potential interaction between the prostaglandin system and UCRP. We suggest that UCRP mRNA may be present constitutively within non-pregnant human endometrium, as revealed by Northern blotting, but at a concentration undetectable by our in-situ hybridization technique, albeit that upregulation accompanies decidualization. In addition, it should be noted that increased concentrations of mRNA may indicate either increased transcription or reduced degradation of the species concerned.
UCRP is specifically upregulated in response to type 1 IFN in several cell and tissue types (Farrell et al., 1979) and its induction parallels initiation of the anti-viral state. The role of UCRP and its conjugate formation are not known, but several functions of UCRP have been demonstrated, including induction of IFN- secretion (Recht et al., 1991) and stimulation of various immune responses (D'Cunha et al., 1996). Interestingly, recent reports indicate that mRNA encoding Ub and a second Ub-related protein may also be upregulated in response to IFN in the porcine uterus (Chwetzoff and d'Andrea, 1997), suggesting that this family of proteins may have an important role in reproduction.
The signal promoting UCRP upregulation in the human endometrium is not clear since there is no molecule yet identified that is directly analogous to IFN- within the human uterus. Previous work employing a simulated pregnant baboon model, in which decidualization is induced by human chorionic gonadotrophin and steroid implants in the absence of a conceptus (Hild-Petito et al., 1995), showed that UCRP protein is present at a high concentration within the endometrial stroma of these animals (Bebington et al., 1999). This suggests that a trophoblast-derived signal may be less important in the primate than within the cow, but further investigation is clearly required into this potentially important mechanism.
In summary, we have demonstrated that decidualization is associated with an increase in detectable concentrations of UCRP mRNA in endometrial stromal cells, further supporting an important role for this protein in implantation and early pregnancy.
Acknowledgments
The authors would like to thank staff in the Pathology Department of Queen's Medical Centre, Nottingham for their help in obtaining tissues, Paul Kells for his assistance with probe generation, and Sam Gray for help with Northern blot protocols and for generation of GAPDH probe. Grant support: C.B. was supported by a University of Nottingham Research Studentship awarded to S.D.F., also by Nottingham University Research and Treatment Unit in Reproduction and Westmead Fertility Centre.
Notes
4 To whom correspondence should be addressed 
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Submitted on February 25, 1999; accepted on July 7, 1999.
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