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a Department of Obstetrics and Gynaecology, School of Human Development and
b Molecular and Cell Biology Section, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom
c Department of Obstetrics and Gynaecology, University of Leicester, Leicester, LE2 7LX, United Kingdom
d Department of Obstetrics/Gynecology, University of Illinois at Chicago, Chicago, Illinois 60612-7313
e Department of Obstetrics and Gynecology, University of Sydney, Westmead Hospital, Sydney, NSW 2145, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-carboxyl group of the C-terminal glycine of ubiquitin and the 
-amino group of a lysine side chain in the target protein. Furthermore, ubiquitin-ubiquitin isopeptide bonds are formed between the C-terminal carboxyl group of ubiquitin and the amino group of primarily lysine 48 in the previous ubiquitin moiety to generate multi-ubiquitin chains [4]. It appears that attachment of multi-ubiquitin chains is required to target a protein for proteolysis by the proteasome. While a growing number of key regulatory proteins including p53 [2, 5], cyclins (reviewed in [2, 6]), transcription factors, and their regulators [7–9]) are degraded by the 26S proteasome after ubiquitylation, it is also clear that in contrast, many cell surface proteins, including some receptor tyrosine kinases [10] and transporters [11], are internalized and degraded in the lysosome system following ubiquitylation. In addition, the rat uterine estrogen receptor is ubiquitylated in response to estradiol [12]. Therefore, ubiquitylation may control targeting of proteins for a number of different fates in the cell.
A protein similar, but not identical, to a head-to-tail ubiquitin-ubiquitin polyprotein, which is recognized by anti-ubiquitin antibodies (ubiquitin cross-reactive protein, UCRP; also known as ISG15), has recently been shown to be secreted by bovine endometrial cells in culture and to be present in bovine uterine flushings [13]. UCRP is up-regulated in human cells in response to interferon (IFN)- and -ß [14–16], and therefore is implicated in the protection of the cell against viral attack. UCRP is generated by the cleavage of a 17-kDa precursor to generate a 15-kDa mature form [17]; this retains the arg-gly-gly sequence at the C-terminus of ubiquitin that is known to be necessary for isopeptide bond formation with lysine side-chain amino groups in target proteins. Indeed, UCRP is found in a conjugated form attached to other intracellular proteins as well as in free monomeric form [18]. The function of this conjugation has not yet been deduced, but it has been suggested that it acts in the removal of viral proteins from the host cell in a manner analogous to that of ubiquitin-dependent protein degradation.
The mammalian endometrium is a complex and dynamic tissue. It is unique in that it is one of the only tissues to undergo cyclic changes and regular regeneration in the adult human, and as such is of great interest to those studying processes of tissue remodeling. The dramatic changes occurring in the stromal cells of the endometrium during the later stages of each secretory phase, which are magnified and continued during the initiation of pregnancy under the influence of the steroid hormones, involve a significant degree of fundamental change to the cell (reviewed in [19]). Ubiquitin is secreted from human term decidual cells in culture [20]. It is possible, therefore, that the ubiquitin system could be involved in the extensive uterine remodeling during the menstrual cycle and pregnancy. Recent work by Hansen and coworkers has shown that UCRP is one of the proteins secreted by the bovine uterus in response to type 1 interferons [21]. The bovine conceptus produces IFN-
during the first 30 days of pregnancy prior to placental attachment, during which time it is thought to maintain the pregnant state [22]. In the early stages of pregnancy, bovine UCRP was found to be secreted by the endometrium, at times coincident with maximal IFN- production by the conceptus [13], and UCRP mRNA in bovine endometrium is up-regulated by IFN- [23].
In this paper we report the presence of ubiquitin and UCRP in the human and baboon endometrium. Changes in levels and distribution of these proteins in the endometrium during the menstrual cycle and pregnancy may provide clues as to their possible specialized roles in this organ.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonpregnant human endometrial specimens were collected from premenopausal women undergoing hysterectomy for indications other than those of malignancy (n = 12). The stage of the 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. Tissue was also obtained from postmenopausal hysterectomy patients (n = 13). Endometrial biopsy tissue from 26 oocyte donors at Days 4 and 10 post-LH surge of a monitored (nontreatment) cycle was supplied by Dr. G. Ndukwe at Queen's Medical Centre, Nottingham, UK.
Human first-trimester decidua was obtained by dissection from the products of conception following therapeutic terminations of pregnancy at the Queen's Medical Centre (n = 12, gestation 7–12 wk). The duration of pregnancy was calculated from the date of the last menstrual period. A rare second-trimester hysterotomy specimen (from a therapeutic termination of pregnancy) was also studied (n = 1). Paraffin-embedded sections of baboon uterus from pregnant, pseudopregnant, and cycling animals were obtained as previously described [24, 25]. Additional tissues from pregnant baboons were kindly provided by Dr. Bambra (Institute for Primate Research, Nairobi, Kenya). All human tissue was obtained with the approval of the Ethics Committee of the Queen's Medical Centre, Nottingham. Animal investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction, and the studies were approved by the Animal Care committees at the University of Illinois at Chicago and the Institute for Primate Research, Nairobi.
All human tissues were collected into fixative (formal saline) for immunohistochemistry within 30 min of removal. Samples for immunoblotting were snap-frozen in liquid nitrogen and stored at -20°C prior to processing. Baboon tissues were fixed in Bouin's solution at room temperature for 24 h.
Antibodies and Proteins
Anti-ubiquitin either was obtained from Dako Ltd. (High Wycombe, UK) or was the generous gift of Prof. R.J. Mayer (School of Biomedical Sciences, Nottingham University, UK). Both anti-ubiquitin antisera were raised in rabbits using the same procedure, and they preferentially recognize ubiquitin-protein conjugates rather than free ubiquitin monomer [26]. Affinity-purified antibody to recombinant human UCRP [27] was the generous gift of Prof. A. Haas (Dept. Biochemistry, Medical College of Wisconsin, Milwaukee, WI). Mouse monoclonal antiserum to insulin-like growth factor-binding protein-1 (IGFBP-1) has been described previously [28]. A mixture of mouse monoclonal antibodies (2B11 and PD7/26) to CD45 were obtained from Dako. Secondary antibodies coupled to biotin- and avidin-biotinylated horseradish peroxidase were obtained from Vector Laboratories (Peterborough, UK). Ubiquitin, from bovine erythrocytes, was obtained from Sigma Chemical Co. (Poole, UK), and recombinant human UCRP was the generous gift of Prof. A. Haas.
Immunohistochemistry
Formaldehyde (4% w:v)- or Bouin's-fixed tissues were dehydrated and embedded in paraffin wax. Sections 4 µm thick were cut, dewaxed in xylene, and rehydrated in a series of ethanols. Endogenous peroxidase was inactivated by treatment of sections with methanol containing 0.6% (v:v) H2O2, and nonspecific staining was prevented by incubation for 1 h in 10% (v:v) nonimmune serum, of the same species as the secondary antibody to be used, diluted in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Sections were then probed for 1 h at room temperature using either rabbit polyclonal antibody to ubiquitin (0.2 µg/ml) or affinity-purified rabbit polyclonal antibody raised against UCRP (10 µg/ml) diluted in blocking solution. Bound primary antibodies were detected using biotinylated goat anti-rabbit IgG antibodies for 30 min followed by incubation with avidin-biotinylated horseradish peroxidase complex for 30 min, all at room temperature. Horseradish peroxidase was visualized with 3,3'-diaminobenzidine and H2O2 using tablets from Sigma. Counterstaining was performed using Harris hematoxylin (BDH, Poole, UK); this was followed by an acid/alcohol wash and several rinses prior to dehydration and mounting in DPX (BDH).
Controls included omission of primary antibody (data not shown) or replacement of primary antibody with nonimmune serum. In addition, absorption controls were performed to test specificity of the anti-ubiquitin and anti-UCRP primary antibodies. Anti-ubiquitin antibody was preincubated overnight at 4°C with ubiquitin (10 µg/ml) prior to probing of sections and was compared to the result for preincubation of antibody with blocking solution alone. Anti-UCRP was preincubated with either ubiquitin (10 µg/ml), recombinant human UCRP (5 µg/ml), or blocking solution overnight at 4°C prior to probing of sections.
A fluorescent double-antibody staining protocol was employed to localize ubiquitin, UCRP, and IGFBP-1 or CD45 on the same tissue section. Sections were incubated with mouse monoclonal antibody raised against IGFBP-1 or CD45 and rabbit antiserum to ubiquitin or UCRP. Bound mouse monoclonal antibody was detected with fluorescein-conjugated horse antibody raised against mouse IgG (Vector) by incubation for 30 min at room temperature. Ubiquitin or UCRP was detected with the antibodies described previously except that bound primary antibody was detected with biotinylated goat antibody to rabbit IgG (Vector) and avidin-Texas Red (Vector), each for 30 min at room temperature. Immunostained sections were mounted in DAPI Antifade (Oncor, Gaithersburg, MD) and viewed with a Leica (Milton Keynes, Bucks, UK) TCS 4D confocal microscope.
Western Blotting
Frozen tissue was rapidly homogenized in electrophoresis sample buffer (62.5 mM 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 mM pepstatin, 4 mM EDTA, 2 mM PMSF, 5 mM N-ethylmaleimide). Five percent (v:v) ß-mercaptoethanol was then added to each sample, and the tubes were 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 [29]. Equal amounts of protein were loaded into each well of a 10- or 15-lane, 12.5% (w:v) acrylamide gel (acrylamide: bis-acrylamide, 37.5:1) surmounted by a 4% (w:v) stacking gel [30]. Estimation of molecular weight was possible due to the concurrent electrophoresis of colored molecular weight markers (Amersham Life Science Ltd., Amersham, UK). Electrophoresis was carried out at 25 mA for approximately 1.5 h followed by overnight electrotransfer of the proteins at 80 mA to nitrocellulose (Hybond C-Super; Amersham Life Science Ltd.) in 25 mM Tris, 192 mM glycine, 20% (v:v) methanol, and 0.1% (w:v) SDS.
After transfer, transfer membranes destined for probing with anti-ubiquitin antibody were heated to enhance immunoblot sensitivity [31]. Nitrocellulose membranes were incubated for at least 1 h with 5% (w:v) milk powder in TBS at room temperature to prevent nonspecific binding of antibodies. Immunostaining was carried out for 2 h at room temperature in blocking buffer that included the previously described polyclonal rabbit serum containing antibodies raised against ubiquitin (0.02 µg/ml) or affinity-purified rabbit antibody raised against UCRP (10 µg/ml). The antiserum was removed, and the transfer was washed repeatedly in TBS containing 0.1% (v:v) Tween 20 (Sigma) before addition of the peroxidase-conjugated secondary antibody (diluted 1:5000 in TBS containing 1% [w:v] milk powder and 0.1% [v:v] Tween 20) and incubation for 1 h. After further washes in TBS/Tween 20, a final rinse was carried out in TBS alone; this was followed by detection of bound peroxidase-conjugated secondary antibodies using an enhanced chemiluminescent system (NEN-DuPont, Stevenage, UK). Hyperfilm-ECL (Amersham Life Science Ltd.) was exposed to the transfer and developed to produce a permanent record of the results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While anti-ubiquitin immunoreactivity in proliferative and early secretory stage gland cells appeared to be cytoplasmic, with little nuclear staining (not shown), immunoreactivity was evident in the nuclei as well as the cytoplasm of gland cells of the late secretory endometrium (Fig. 1b). Sections obtained from a second-trimester hysterotomy demonstrated immunoreactivity within the decidua parietalis (Fig. 1f) that was similar in intensity to that for the first trimester. No endometrial immunoreactivity was observed in any of the postmenopausal women, although some staining was seen within the myometrial region (results not shown).
Confirmation of the presence of ubiquitin-protein conjugates in endometrium was provided by immunoblotting of electrophoretograms of fresh tissue using anti-ubiquitin antibody. Little or no free ubiquitin (molecular mass 8.5 kDa) was detected in the tissue, but a large number of immunoreactive high molecular mass polypeptides were present, forming a characteristic high molecular mass "smear" (Fig. 2, lanes 1–12). Despite the appearance of anti-ubiquitin immunoreactivity in decidual cells during pregnancy (Fig. 1, c, e, and f vs. a and b), no obvious differences between the levels or pattern of ubiquitin-protein conjugates with a molecular mass greater than 21 kDa could be observed between nonpregnant and pregnant tissue (Fig. 2, lanes 1–6 vs. lanes 7–12), although there did appear to be lower levels of ubiquitin-protein conjugates of less than 21 kDa in pregnant tissue. The appearance of new decidual ubiquitin-protein conjugates during pregnancy might have been obscured on immunoblots by high levels of ubiquitin-protein conjugates present in the epithelial cells of nonpregnant and pregnant tissue. In addition to ubiquitin, anti-ubiquitin antibody may detect proteins similar to ubiquitin, and indeed is known to detect UCRP in tissue sections, but may be less sensitive to UCRP or UCRP-conjugated proteins on immunoblots. No polypeptides were visualized when nonimmune rabbit antiserum was substituted for anti-ubiquitin antiserum in the blotting procedure (results not shown).
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To clarify whether or not the anti-ubiquitin antibody detects ubiquitin or UCRP in sections of endometrium, sections were probed with an anti-recombinant human UCRP rabbit polyclonal antibody [27]. In contrast to the pattern of immunoreactivity seen with anti-ubiquitin antibody, anti-UCRP immunoreactivity was absent from epithelial gland cells of nonpregnant tissue (Fig. 3, a and b, vs. Fig. 1, a and b; see arrow in Fig. 1a). This would seem to indicate that the anti-UCRP antibody employed does not detect ubiquitin in paraffin-embedded endometrial sections and that anti-ubiquitin immunoreactivity in the gland cells truly reflects the presence of ubiquitin alone. Anti-UCRP immunoreactivity was present in the decidual cells of pregnant tissue during the first (Fig. 3, c and d) and second trimester (Fig. 3, f and g), in a pattern similar to that for anti-ubiquitin immunoreactivity in these tissues (Fig. 1, c, e, and f, respectively). Incubation of anti-UCRP antibody with recombinant human UCRP (5 µg/ml antibody) prior to application to sections abolished immunoreactivity (Fig. 3e). In contrast, preincubation of anti-UCRP antibody with ubiquitin (10 µg/ml antibody) did not significantly alter the levels of anti-UCRP immunoreactivity seen in tissue sections (Fig. 3d). These controls suggest that anti-UCRP immunoreactivity in tissue sections specifically indicates the presence of UCRP. Insignificant immunoreactivity was observed in fetal membranes (data not shown).
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Anti-UCRP antibody detected several conjugates (> 40 kDa) in every example of decidua examined by immunoblotting, including an apparently abundant species of approximately 40 kDa; but no free UCRP was detectable (Fig. 4b, lanes 1–6). In contrast, only a single anti-UCRP-immunoreactive polypeptide of 40 kDa was detectable in some of the nonpregnant endometrial tissues (Fig. 4a, lanes 1–6). Human recombinant UCRP, applied to the electrophoresis gel, was detected by anti-UCRP antibody as expected; but ubiquitin was not recognized (data not shown), indicating that the polypeptides detected by antibody to UCRP were conjugates of intracellular proteins to UCRP and not to ubiquitin. Replacing anti-UCRP antibody with nonimmune rabbit serum abolished all immunoreactivity on immunoblots (not shown).
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Decidual cells secrete IGFBP-1 [32] and can therefore be readily identified with an antibody to IGFBP-1. Pregnant endometrium simultaneously probed with mouse monoclonal anti-IGFBP-1 and antibody to ubiquitin exhibited IGFBP-1 immunoreactivity in what appeared to be perinuclear vesicles (Fig. 5a, arrow labeled "v"). The nucleus of the same cell exhibited anti-ubiquitin antibody immunoreactivity (Fig. 5b, arrow labeled "n"). Anti-ubiquitin immunoreactivity was also visible in cells with much lower levels of IGFBP-1, and indeed the intensity of anti-ubiquitin antibody immunoreactivity did not directly correlate with the apparent cellular content of IGFBP-1. Anti-UCRP antibody immunoreactivity (Fig. 5d, arrow) was present in cells also immunoreactive to anti-IGFBP-1 (Fig. 5c, arrow). Anti-UCRP antibody immunoreactivity appeared to be distributed in a granular fashion throughout the cell (Fig. 5d, arrow). Endometrium contained not only decidual cells but also cells derived from the bone marrow. We used antibody to CD45 to localize bone marrow-derived cells [33]. Anti-ubiquitin (Fig. 5f) and anti-UCRP antibody immunoreactivity (Fig. 5h) were both clearly present in cells negative for CD45 (Fig. 5, e and g) and typical in morphology to decidua compacta (Fig. 5, f and h, arrow labeled "dc"). However, anti-UCRP immunoreactivity was also present in anti-CD45 antibody-immunoreactive cells (Fig. 5h for UCRP; Fig. 5g for CD45, arrow labeled "bm"). Therefore it appears that UCRP is present in both decidual cells and bone marrow-derived cells of the pregnant endometrium.
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The study of baboon tissues allowed us to observe protein distribution in tissue that is not available from humans for practical or ethical reasons. A range of tissues were examined, including those from cycling animals from the follicular (n = 3) and luteal (n = 3) phases. Tissues from pregnant animals were also studied (n = 6). This allowed us to collect decidua from as early as Day 17 of pregnancy and to compare maternal tissues collected from the site of implantation with that from other regions of the uterus at that time. The true decidual reaction in the baboon usually occurs later in pregnancy than in the human, and only in response to a conceptus [34]. Study of specimens from later in pregnancy was therefore important. Tissue was also available from an animal treated with hCG to mimic pregnancy in the absence of invading trophoblast [25]. In contrast to observations in the human tissues (Fig. 1, a and b), there was some anti-ubiquitin immunoreactivity in the stroma of the endometrium from Day 13 after ovulation (Fig. 6a), but no intense staining was observed until Day 71 of pregnancy (Fig. 6b), increasing into the later specimens (decidua from Day 176 of pregnancy showed clear immunoreactivity, results not shown). Interestingly, the hCG-treated animal also displayed significant stromal staining (Fig. 6d), suggesting that, in the baboon at least, this is an important hormonal factor affecting the presence of anti-ubiquitin immunoreactivity, and hence probably ubiquitin and UCRP-protein conjugate formation, in this tissue. Anti-UCRP immunoreactivity was present in the decidua of Day 71 pregnant endometrium (Fig. 6c) although at a lower intensity compared to that in human decidua, possibly reflecting lower affinity of anti-recombinant human UCRP for baboon UCRP or lower levels in baboon tissue of UCRP compared to ubiquitin. Replacing antibody with nonimmune rabbit serum abolished immunoreactivity (Fig. 6e).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Taking into account the specificity of antibody to ubiquitin used in this study, the results we obtained indicate that ubiquitin-protein conjugates are present in the epithelial gland cells in nonpregnant and pregnant humans and baboons. Free ubiquitin is presumably also present in these cells. Free ubiquitin has been detected in the uterine flushings of both nonpregnant and pregnant cows, and it is possible that ubiquitin is released from epithelial gland cells into the lumen of the gland. Anti-ubiquitin immunoreactivity is largely absent from nonpregnant stroma but appears in the decidual cells of pregnant tissue. In some cases, anti-ubiquitin immunoreactivity is seen in the region of stroma underlying the luminal epithelium of nonpregnant women (Fig. 1a, labeled "p"). Interestingly, these tissues were taken from women in the late secretory phase of the menstrual cycle and may represent cells undergoing pseudo-decidualization; together with the observations of immunoreactivity in pregnant tissue (Fig. 1, c, e, and f), the results indicate that decidualization is accompanied by an increase in anti-ubiquitin immunoreactivity. The pattern of anti-ubiquitin-immunoreactive polypeptides after electrophoresis and immunoblotting (Fig. 2) of nonpregnant and pregnant human endometrium probably represents the presence of many different polypeptides conjugated to multi-ubiquitin chains of different sizes. The absence of an immunoreactive band corresponding to ubiquitin monomer may simply reflect the lack of sensitivity of this antibody to the free form of ubiquitin [26] and the relatively low levels of free ubiquitin in the tissue. Anti-ubiquitin immunoreactivity may be the result of the presence of ubiquitin, UCRP, or ubiquitin and UCRP together. Human decidual cells are immunoreactive to the specific anti-UCRP antibody (Fig. 3), indicating that at least some of the anti-ubiquitin immunoreactivity seen in sections of human tissue containing decidual cells is due to the presence of UCRP. These novel results suggest that UCRP has a role in decidualization or in the function of the decidual cell. Anti-ubiquitin antibody immunoreactivity in decidual cells is stronger within the nucleus than in the cytoplasm (Fig. 1, e and f), this pattern being reversed with anti-UCRP antibody (Fig. 3g), which suggests that nuclear immunoreactivity in decidual cells may be largely due to the presence of ubiquitin alone. The absence of immunoreactivity from postmenopausal endometrium would further indicate a role for this system in the activities of this dynamic tissue. We cannot exclude the possibility that UCRP is present in the stroma or epithelial gland cells of nonpregnant tissue, as it is possible that the concentrations are too low to be detected by the antibody employed. Indeed, UCRP appears to be expressed constitutively at low levels in several cell types [18]. The pattern of polypeptides immunoreactive to antibody to UCRP following electrophoresis and immunoblotting of stromal and decidual tissue suggests that decidualization is accompanied by appearance of greater levels of a 40-kDa UCRP conjugate and new higher molecular mass species of UCRP-protein conjugates (Fig. 4). In addition, the pattern of UCRP-protein conjugates in pregnant tissue (Fig. 4b, lanes 1–6) is very different from that of ubiquitin-protein conjugates seen in pregnant and nonpregnant tissue (Fig. 2, lanes 1–12). Ubiquitin appears to exist in a far greater number of conjugated species as compared to UCRP. This may reflect a greater number of polypeptide targets for ubiquitylation as compared to modification by UCRP and may indicate different cellular roles for these related proteins. The results from immunoblots again suggest that UCRP-protein conjugates are largely confined to decidual cells. Therefore, we conclude that UCRP, certainly in the conjugated form, is up-regulated in the decidua during pregnancy. Anti-UCRP immunoreactivity also appears in the baboon endometrium accompanying decidualization during pregnancy or induced by hCG. The appearance of anti-UCRP immunoreactivity in decidual cells at the beginning of pregnancy indicates that it may play an important role in the establishment of the conceptus. An anti-UCRP-immunoreactive polypeptide of 16 kDa, which appears to be free UCRP [13], is found in uterine washes of pregnant, but not nonpregnant, cows and is one of a small number of proteins released from the endometrium in response to IFN [21], which suggests that the protein is specifically secreted from uterine tissue. The functional significance of these findings is not clear, although the secretion of IFN-
by monocytes in response to UCRP has previously been reported [37] and the protein therefore may play a vital role in coordination of the complex network of IFNs during the initiation of bovine pregnancy.
We have localized anti-UCRP-immunoreactive protein to decidual cells that have high levels of secretory activity and that contain, and are known to secrete, IGFBP-1. UCRP appears also to be present in cells containing CD45. However, we do not know what the relationship may be between the appearance of UCRP-conjugates and the release of free UCRP from the endometrium. For ethical reasons it is less easy to obtain human uterine flushings to test whether, as in the cow, UCRP is present. It will be of great interest to determine whether UCRP in decidual cells is localized to the secretory apparatus. We have employed qualitative methods of immunohistochemistry and immunoblotting to study changes in levels and distribution of ubiquitin and UCRP within the endometrium of humans and baboons during pregnancy and, even allowing for the limitations of these methods, the perceived changes appear striking.
The roles of UCRP and its conjugates are not known. UCRP has been detected by immunolocalization in several human tissues [27, 38] and has been hypothesized to play a part in anchoring soluble proteins to the cytoskeleton [27]. UCRP is also thought to play a part in protection of the cell against viral infection, and it is up-regulated by IFN- in bovine endometrium [13, 21] and by IFN- and -ß in human cells of various types [15, 35, 37]. The processes occurring in the endometrium at the initiation of pregnancy have often been compared to an inflammatory response, and this up-regulation of UCRP may further support this analogy. It has been postulated that UCRP regulates secretion of IFN- [37]. As such it may coordinate the activity of these two branches of the IFN family and provide important signaling activity in the uterus at the time of implantation. It is unknown whether conjugation of UCRP to proteins targets them for degradation by the 26S proteasome in the same way as ubiquitin conjugation. Further studies will be required to elucidate the role of UCRP in the human endometrium during pregnancy, but the dramatic up-regulation described in this report indicates an important function for UCRP in gestation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 44 115 970 9969; fergus.doherty{at}nottingham.ac.uk
Accepted: November 17, 1998.
Received: September 25, 1998.
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J. K. Pru, K. J. Austin, D. J. Perry, A. M. Nighswonger, and T. R. Hansen Production, Purification, and Carboxy-Terminal Sequencing of Bioactive Recombinant Bovine Interferon-Stimulated Gene Product 17 Biol Reprod, August 1, 2000; 63(2): 619 - 628. [Abstract] [Full Text]
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C. Bebington, F.J. Doherty, G. Ndukwe, and S.D. Fleming The progesterone receptor and ubiquitin are differentially regulated within the endometrial glands of the natural and stimulated cycle Mol. Hum. Reprod., March 1, 2000; 6(3): 264 - 268. [Abstract] [Full Text] [PDF]
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C. Bebington, F.J. Doherty, and S.D. Fleming Ubiquitin cross-reactive protein gene expression is increased in decidualized endometrial stromal cells at the initiation of pregnancy Mol. Hum. Reprod., October 1, 1999; 5(10): 966 - 972. [Abstract] [Full Text] [PDF]
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