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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

October 2000
Volume IV, Number 4
ISSN: 1537-6583
Pages: 240-252

Ubiquitin And Ubiquitin-Protein Conjugates Are Present In Human Cytotrophoblast Throughout Gestation

Bebington, C^1,4., Doherty, F.J²., Fleming, S.D^3,5.

School of Human Development ¹ and School of Biomedical Sciences ², University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, U.K. and Department of Obstetrics & Gynaecology, University of Sydney, Westmead Hospital, Sydney, NSW 2145, Australia³.  ⁴Current address: Sheffield IVF Centre, Sheffield, S7 1RA, UK.  ⁵To whom correspondence should be addressed.

Running Title: Ubiquitin in human placenta

Key Words: human/ placenta/ pregnancy/ ubiquitin/ UCRP

Grant support: CB was supported by a University of Nottingham Research Studentship awarded to SDF, also by Nottingham University Research and Treatment Unit in Reproduction and Westmead Fertility Centre

Ethics: All tissue was obtained with the approval of the Ethics Committee of Queen's Medical Centre, Nottingham

Acknowledgments: The authors would like to thank all those who were involved with tissue collection, particularly Andrea Cooper, Steve Smith and Graham Robinson of the University of Nottingham, UK

Summary

Ubiquitin is a small protein involved in many intracellular processes. We have previously shown that levels of ubiquitin change during the process of decidualisation in the human uterus at the beginning of pregnancy. Other workers have shown that the ubiquitin system may be essential for normal murine placental development. In this investigation we employed immunohistochemistry and immunoblotting techniques to study the distribution and abundance of ubiquitin and ubiquitin-protein conjugates within human placental specimens from throughout gestation. Trophoblast from two pathological conditions, ectopic pregnancy and pregnancy-induced hypertension (PIH), was also investigated. Ubiquitin was detected within both the cytoplasm and nucleus of the cytotrophoblast layer only. Both monomeric and conjugated forms of ubiquitin were detected. The relative abundance of ubiquitin did not change through gestation or in the two disorders of pregnancy studied. Ubiquitin cross-reactive protein was not detected in the tissues of interest. This is the first report to demonstrate the cell-specific localisation of ubiquitin and ubiquitin-protein conjugates in the human cytotrophoblast and provides supportive evidence that ubiquitin may be important during placental development.

Introduction

Ubiquitin is a small, highly conserved protein that appears to be involved in a number of intracellular processes. The major role for ubiquitin appears to be in the covalent marking of intracellular proteins, especially short-lived regulatory proteins, for proteolysis (reviewed in Weissman,1997). Following covalent addition of ubiquitin (ubiquitylation), proteins are degraded by a large cytosolic protease known as the 26S proteasome (Hough et al,, 1987). Ubiquitylation of proteins consists of the formation of an isopeptide bond between the a-carboxyl group of the C-terminal glycine of ubiquitin and the e-amino group of a lysine side chain in the target protein. Additional 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 (Chau et al, 1998). Attachment of ubiquitin to target proteins is a multi-step process involving three classes of enzymes termed ubiquitin-activating (Ub-A or E1 enzymes), ubiquitin-conjugating (Ub-C or E2 enzymes) and ubiquitin-protein ligating (Ub-L or E3 enzymes). Substrate recognition may require specific E2 or E3 enzymes (Weissman, 1997).

Several proteins are known substrates for multi-ubiquitylation and subsequent degradation by the 26S proteasome, including regulatory proteins such as p53 (Chowdary et al, 1994), cyclins (Glotzer et al, 1991), transcription factors and their regulators (Alkalay et al, 1995; Stancovski et al, 1995; Traenckner and Baeuerle, 1995). It is also clear that although some cell surface proteins, including some receptor tyrosine kinases (Mori et al, 1993) and transporters (Roth and Davis, 1996), are ubiquitylated, they are degraded in the lysosome rather than via the ubiquitin-dependent proteasome. Furthermore, ubiquitylation may be necessary for the ligand-mediated down-regulation of certain steroid hormone receptors including the rat and human oestrogen receptor (Nawaz  et al,  1999; Nirmala and Thampan, 1995) and the chicken and human progesterone receptor (Syvälä et al, Bebington et al, 2000b). In contrast, some proteins such as the histones (Goldknopf and Busch, 1975) are subject to ubiquitylation but are not degraded. Therefore, ubiquitylation may control targeting of proteins for a number of different fates in the cell.

The ubiquitin-mediated degradation of proteins may play an important role in reproductive processes involving tissue remodelling and development (reviewed in Bebington et al, 2000a). Of particular interest in the field of reproductive physiology is the finding that an E2 enzyme is necessary for normal placental development in the mouse (Harbers et al, 1996). These workers demonstrated that embryos lacking the UbcM4 gene (encoding an E2 enzyme) suffered intra-uterine growth retardation and perinatal death. This enzyme is homologous with the human gene UbcH7, which also encodes a functional E2 enzyme (Nuber et al, 1996). The only organ to show consistent pathological defects in homozygous mutant animals was the placenta, specifically the foetal mesenchyme in the labyrinth layer and the placental vasculature (Harbers et al,1996). A number of homozygous mutant embryos died around day 11.5 of gestation, a time when the placenta becomes vital for intrauterine survival. This evidence suggests that activity of the ubiquitin system is necessary for normal placental growth and development.

Several developmentally-regulated genes of Drosophila melanogaster play a role in ubiquitylation, including the fat facets gene which is involved in eye development and encodes a de-ubiquitylating enzyme (Huang et al,1995)and the hyperplastic disk gene, encoding an E3 enzyme, which appears to play a major role in development and reproductive viability (Mansfield et al, 1994). The ubiquitin system has also been implicated in developmental processes in Dictyostelium discoides (Clark et al, 1997; Lindsey et al, 1998) and Caenorhabditis elegans (Zhen et al,1997) and during chick embryogenesis (Smith-Thomas et al,1994) and human myogenesis (Bornman et al, 1996). Developmental processes requiring whole-scale destruction of organelles are often accompanied by increased protein ubiquitylation; for example the development of the chick lens (Scotting et al, 1991) and the plant vascular system (Bachmair et al,1990).

A protein similar, but not identical, to a head to tail ubiquitin-ubiquitin poly-protein (ubiquitin cross-reactive protein, UCRP, also known as ISG15; Haas et al, 1987), which is recognised by anti-ubiquitin antibodies, has recently been shown to be secreted by bovine endometrial cells in culture and to be present in bovine uterine flushings (Austin et al,1996). We have previously detected UCRP mRNA and protein in decidualised endometrial stromal cells (Bebington et al,1999a; 1999b).

The mammalian placenta is a temporary organ necessary for physiological exchange between maternal and foetal systems during intrauterine gestation. It exhibits profound changes in size and nature during pregnancy, first implanting into the uterine lining and then dramatically increasing in size during the first half of pregnancy. Despite the indications that the ubiquitin system is involved in numerous examples of tissue remodelling and development, and specifically in placental development, there has not previously been an investigation into levels of placental ubiquitin. We were interested to see whether ubiquitin or UCRP were present within human trophoblast tissues during different stages of gestation, or in pathological conditions of pregnancy. To investigate this possibility, we employed immunohistochemical and western blotting techniques.

Materials and Methods

Sample Collection
Placental bed biopsies were obtained from therapeutic terminations of pregnancy in the first (n=10, immunohistochemistry; n=2, western blotting) or second trimester (n=3, immunohistochemistry; n=1, western blotting) and from placenta delivered by caesarean section at term in uncomplicated pregnancies (n=13, immunohistochemistry; n=2, western blotting). We also used immunohistochemistry to study placenta delivered by caesarean section at term where there were indications of pregnancy-induced hypertension (PIH; n=6) and intra-fallopian trophoblast from ectopic pregnancies (n=10). Tissues were a generous gift of A. Cooper (University of Nottingham, UK).

Criteria for inclusion in the PIH category included:

• diastolic blood pressure exceeding 90mmHg
• systolic blood pressure exceeding 140mmHg
• previously normotensive
• proteinuria in excess of 300mg/l in a 24-hour collection, or ‘two pluses’ on dipstick testing of a random sample.

All human tissues were collected into 10% (w/v) formal saline or 4% (w/v) paraformaldehyde for immunohistochemistry immediately after surgery. Samples for immunoblotting were snap-frozen in liquid nitrogen and stored at -20^oC prior to processing. All work was approved by the Ethics Committee of Queen’s Medical Centre, Nottingham.

Antibodies
Anti-ubiquitin was either obtained from DAKO Ltd (High Wycombe, UK) or was the generous gift of Prof. R.J. Mayer, University of Nottingham, U.K. Both anti-ubiquitin antisera were raised in rabbits using the same procedure and preferentially recognise ubiquitin-protein conjugates rather than the free ubiquitin monomer (Haas and Bright, 1985). Affinity purified rabbit polyclonal antibody to recombinant human UCRP (Loeb and Haas, 1994) was the generous gift of Prof. A. Haas, Medical College of Wisconsin, USA. Secondary antibodies coupled to peroxidase or biotin and avidin-biotinylated horseradish peroxidase were obtained from Vector Laboratories, Peterborough, U.K.

Immunohistochemistry
Fixed tissues were dehydrated and embedded in paraffin wax. Sections 4m m thick were cut, de-waxed in xylene, and re-hydrated in a series of ethanols. Endogenous peroxidase was blocked in methanol containing 6% (v/v) H₂O₂, and non-specific staining was prevented by incubation for one hour in 10% (v/v) non-immune serum, of the same species as the secondary antibody to be used, diluted in Tris-buffered saline (TBS, 50mM Tris-HCl, 150mM NaCl, pH 7.5). Sections were then probed using either rabbit primary antibody to ubiquitin (0.2m g/ml) or affinity-purified rabbit polyclonal antibody raised against UCRP (10m g/ml), diluted in blocking solution, for one hour at room temperature. Bound primary antibodies were detected using biotinylated goat anti-rabbit IgG antibodies for 30 minutes at room temperature followed by avidin-biotinylated horseradish peroxidase complex (30 minutes at room temperature). Horseradish peroxidase was visualised with 3,3’ diaminobenzedine and H₂0₂ using tablets from Sigma Chemical Co, Poole, U.K. Counterstaining was performed using Harris’ Haematoxylin (BDH, Poole, UK) followed by an acid/alcohol wash and several rinses prior to dehydration and mounting in DPX (BDH).

Controls used included omission of primary antibody or use of pre-immune rabbit serum in place of primary antibody (data not shown). In addition, absorption controls were performed to test the specificity of the anti-ubiquitin primary antibody. Incubation of ubiquitin (Sigma, UK) with anti-ubiquitin at 10m g/ml antibody at 4° C was performed overnight prior to probing sections and was compared to the result of incubating antibody with blocking solution alone (Fig. 1 panel A versus panel B).

Western Blotting
Frozen tissue was rapidly homogenised in electrophoresis sample buffer (62.5mM Tris-HCl, 2.3% (w/v) SDS, 10% (v/v) glycerol, and 0.01% (w/v) bromophenol blue containing protease inhibitors (3mM pepstatin, 4mM EDTA, 2mM PMSF, 5mM N-ethylmaleimide). 5% (v/v) b -mercaptoethanol was then added to each sample and the tubes were boiled for five minutes 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 550nm (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 Life Science Ltd., Amersham, UK). Electrophoresis was carried out at 25mA for approximately 1.5 hours followed by overnight electro-transfer of the proteins to nitro-cellulose (Hybond C-Super, Amersham Life Science Ltd.) in 25mM Tris, 192mM glycine, 20% (v/v) methanol and 0.1% (w/v) SDS at 80mA.

Following transfer, nitrocellulose membranes were autoclaved in TBS to enhance immunoblot sensitivity (Swerdlow et al, 1986) and were incubated for at least one hour with 5% (w/v) milk powder in TBS at room temperature, to prevent non-specific binding of antibodies. Immunostaining was carried out for two hours at room temperature in blocking buffer containing the previously described polyclonal rabbit serum containing antibodies raised against ubiquitin (0.02m g/ml) for two hours at room temperature. 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 at 1:5000 in TBS/Tween and incubation for one hour. 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, U.K.). Hyperfilm-ECL (Amersham Life Science Ltd.) was exposed to the transfer and developed to produce a permanent record of the results.

Results

Cytotrophoblast cells from throughout gestation demonstrated strong immunolocalisation of ubiquitin while the syncytiotrophoblast layer was consistently negative (Fig. 1, panels A, C and D). The placenta at term demonstrated less overall immunoreactivity since the cytotrophoblast layer was degraded and patchy by this stage of gestation (Fig. 1, panel D). Anti-ubiquitin immunoreactivity within the cytotrophoblast layer was seen in both nuclei and cytoplasm (see Table 1). Cells consistent in morphology and location with Hofbauer cells were not immunoreactive (Fig. 1, panel A, labelled ‘hb’). Endothelial cells within the placental villus were immunoreactive (Fig. 1, panel C, arrowed), particularly within samples obtained at term.

Immunoreactivity was abolished by the pre-absorption of primary antibody with monomeric ubiquitin (Fig. 1, panel B) indicating that staining was due to specific antibody-antigen interactions. No immunoreactivity was seen when non-immune rabbit serum was substituted for anti-ubiquitin in the immunohistochemistry or western blotting procedures (results not shown). In addition to ubiquitin, anti-ubiquitin antibody may also detect proteins similar to ubiquitin, and indeed is known to detect UCRP in tissue sections. However, no immunoreactivity was detected when tissue sections were probed with antibody to UCRP (results not shown), indicating that immunoreactivity was indeed due to the presence of ubiquitin alone.

There was little apparent difference between trophoblast from uncomplicated term pregnancy or that complicated by PIH (Fig. 1, panel D versus panel E), or between trophoblast from the first trimester of pregnancy from uterine or ectopic sites of implantation (Fig.1, panel A versus panel F).

Placental bed biopsies from throughout gestation were also studied by immuno-blotting. Immunoreactivity was seen with anti-ubiquitin within the high molecular-weight region of the gel (Fig. 2) in a typical ‘smear’, indicating the presence of ubiquitin-protein conjugates. These conjugates did not appear to vary in abundance throughout gestation. A band co-migrating with free ubiquitin standard (Fig. 2, molecular mass 8.5kDa, labelled ‘Ub’) was also detected, and may represent free ubiquitin within these tissues. Intensity of this band did not vary between gestational ages.

Discussion

We have previously reported that ubiquitin and UCRP are present in the maternal portion of the human placenta (Bebington et al, 1999a). Previous studies by other workers have shown that functions of the ubiquitin system are vital in normal placental development in the mouse (Harbers et al, 1996). In this paper we report the presence of ubiquitin in the human cytotrophoblast throughout gestation, in ectopic pregnancy, and in pregnancies complicated by PIH.

We have demonstrated that both monomeric ubiquitin and ubiquitin-protein conjugates are present in the placental bed. Anti-ubiquitin immunoreactivity is largely absent from the syncytiotrophoblast layer and from the Hofbauer cells of the placental villus (Fig. 1). The pattern of anti-ubiquitin immunoreactive polypeptides observed after electrophoresis and immuno-blotting of placental bed samples (Fig. 2) probably represents the presence of many different polypeptides conjugated to multi-ubiquitin chains of different sizes. Although the antibody employed preferentially recognises ubiquitin in the conjugated form (Haas and Bright, 1985) we also detected a band corresponding in size to monomeric ubiquitin (Fig. 2, labelled ‘Ub’) which presumably indicates a relatively high level of free ubiquitin within these tissues.

We did not detect any immunoreactivity when a primary antibody specific to UCRP was employed to probe these tissue sections (results not shown). We cannot exclude the possibility that UCRP is present at concentrations too low to be detected by the techniques employed. However, since strong staining was seen within other tissues using similar protocols (Bebington et al, 1999a), it is likely that all anti-ubiquitin immunoreactivity indeed represents the presence of ubiquitin itself. UCRP has previously been shown to be present in uterine flushings of the cow, apparently due to secretion by bovine endometrial cells in response to trophoblast-derived interferon-t (Austin et al, 1996). In the human, decidualised endometrial stromal cells also contain UCRP (Bebington et al, 1999a). From the findings of this investigation it does not appear likely that conceptus-derived tissues contribute to levels of uterine UCRP.

Abnormal placental development is often seen in patients suffering from PIH (Khong and Sawyer, 1991). We did not observe any changes in the abundance or distribution of ubiquitin in placentae from patients with PIH in comparison to those from normal pregnancies. This is somewhat surprising since other workers have indicated that the ubiquitin system may be essential for normal placental growth (Harbers et al, 1996). The fact that no changes were seen in the abundance of ubiquitin in this condition does not mean that the ubiquitin system is not involved in the pathogenesis of PIH. It is possible that the concentration of monomeric ubiquitin is not the rate-limiting step in protein ubiquitylation within the placenta, and that other stages of the multi-step pathway towards protein degradation are altered in PIH. The immuno-blotting of placental biopsies from patients with PIH would allow a comparison of the total levels of ubiquitin-protein conjugates in this tissue and would allow us to determine whether specific changes may be detected.

The function of the ubiquitin system in the placenta is not known. The abundance of ubiquitin within the rapidly-dividing cytotrophoblast but not within the fully differentiated syncytiotrophoblast tissues, suggests that it may be involved in tissue growth and differentiation. Previous workers have demonstrated that ubiquitin is involved in cell-cycle progression (Glotzer et al, 1991) and in some examples of tissue remodelling (Murdoch et al,1996; Scotting et al, 1991). The E2 enzyme shown to be necessary for placental growth (Harbers et al, 1996) is thought to be involved in the ubiquitylation of p53 (Nuber et al, 1996), but there is no evidence that p53 accumulates in the placenta of homozygous mutant embryos. The ubiquitin system is multi-functional and it may be that a number of processes employ ubiquitylation at some point during placental development.

In summary, this investigation is the first to localise ubiquitin and to demonstrate the presence of ubiquitin-protein conjugates in human trophoblast during pregnancy. Further work is clearly required to elucidate the role of the ubiquitin system during this vital process.

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Table 1
Result of probing sections of tissue with anti-ubiquitin

Tissue sections were prepared and probed with antibody to ubiquitin as described in Materials and Methods. Intensity of immunostaining was graded on a three-point scale from ‘-’ (negative) to ‘++’ (strongly positive).

Figure 1
Anti-Ubiquitin immunoreactivity in the human placental bed

Tissue were obtained, processed and probed with anti-ubiquitin except panel B, probed with anit-ubiquitin pre-incubated with ubiquitin, as described in Materials and Methods.

Specimens shown include trophoblastic villi from first (panels A and B) or second (panel C) trimester terminations of pregnancy. Panel D shows tissue obtained from an elective caesarean term delivery, while panel E shows tissue obtained from a patient delivered by caesarean section near to term due to PIH. Panel F demonstrates a section obtained from a tubal implantation site removed during the early stages of ectopic pregnancy.

Labels indicae: 'ctb', cytotrophoblast; 'stb', syncytiotrophoblast; 'hb' Hofbauer cells. Arrow indicates blood vesels. Scale bar equals 50mm and applies to all panels.

Figure 2
Anti-Ubiquitin immunoreactive polypeptides in placental samples throughout gestation

Placental bed tissues were obtained, processed an probed with rabbit polyclonal anti-ubiquitin as described in Materials and Methods.

Lanes 1 and 2, placenta from the first trimester of pregnancy; lane 3, placenta from the second trimester of pregnancy; lanes 4 and 5, placenta from uncomplicated term delivery. Positions of molecular weight standards and of monomeric ubiquitin are indicated.