Differential Expression of Genes in the Endometrium at ... - BioOne

BIOLOGY OF REPRODUCTION 70, 406–414 (2004) Published online before print 15 October 2003. DOI 10.1095/biolreprod.103.020719

Differential Expression of Genes in the Endometrium at Implantation: Upregulation of a Novel Member of the E2 Class of Ubiquitin-Conjugating Enzymes1 Michael H. Melner,2–4 Nicole A. Ducharme,3,4 Alan R. Brash,5 Virginia P. Winfrey,4 and Gary E. Olson4 Departments of Obstetrics and Gynecology,3 Cell Biology,4 and Pharmacology,5 Vanderbilt University School of Medicine, Nashville, Tennessee 37232 ABSTRACT

stage. This uterine receptivity is principally controlled by the differentiation of the endometrium from the proliferative to the secretory phases, effected by the changing levels of estradiol during the proliferative phase and increasing levels of progesterone during the secretory phase of the cycle [1]. Following attachment of the embryo, a second series of events is initiated, specifically at the implantation sites, which controls the differentiation of the endometrium into a highly specialized tissue for molecular communication with the embryo and for the provision of nutrients and exchange of metabolites that sustain the embryo during pregnancy. This second series of events is not well characterized and the genes controlling endometrial differentiation at this stage are only beginning to be identified and examined. In particular, the genes induced by the embryo, specifically at the implantation site by embryo attachment, are of particular importance to the success of pregnancy. Epithelial cells play a critical role in implantation, both during initial embryo attachment and its subsequent invasion [2]. During the attachment phase, endometrial epithelial cell-surface proteins, including integrins and sulfated oligosaccharide selectin ligands, may function in initial adhesion of the embryo [3–8]. Subsequently, epithelial cells undergo dramatic structural and functional changes in their interaction with the trophoblast and its invasion through the basement membrane [9–11]. Understanding the cellular and functional changes in epithelial cells, induced by implantation, will provide clues to the mechanisms underlying this process as well as potential pathological changes preventing implantation. Recent studies have aimed at identifying differentially expressed genes associated with the implantation process. For genes potentially involved in development of the receptive endometrium, microarrays were used to probe for changes in the expression of genes in human endometrial biopsies from the secretory phase, when progesterone is high, compared with the proliferative phase, when estradiol is high [12]. In these screens, certain genes demonstrate prominent changes in expression during the secretory phase with upregulation of the ApoE, phospholipase A2, glucuronyltransferase I, pregnancy-associated endometrial alpha 2-globulin, mammaglobin, and dickkopf-1 genes, and downregulation of the intestinal trefoil factor, frizzled related protein, matrilysin, dipeptidyl aminopeptidase like protein, G-protein receptor kinase 5, HM145, and a-1 type XVI collagen genes. These studies suggest increased levels of cholesterol/lipid trafficking proteins, secretory glycoproteins, and signaling molecules during the receptive phase of the cycle and decreased expression of genes involved in cellular maintenance/repair and for secreted proteases. Studies were also performed in the mouse comparing implantation and interimplantation uteri [13]. Interestingly, this study noted a reduction in the expression of a number

The process of embryo attachment and implantation is accompanied by dramatic cellular and functional changes in the endometrium, the control and mechanisms of which are not clearly understood. The cDNA cloning of differentially expressed genes, specifically at implantation sites in the rabbit endometrium, was used to identify genes controlling functional and remodeling changes. Tissue from the endometrium of Day 6¾ (preimplantation) and Day 8 (implantation initiation) pregnant rabbits was used to screen for differentially expressed genes by combined cDNA subtraction/suppressive hybridization. Twentynine differentially expressed genes were identified encoding protein modification enzymes, signaling proteins, structural proteins, and enzymes. One of these is a novel member of the E2 ubiquitin-conjugating enzyme family we have designated UBCi (i for implantation), which displayed dramatic nucleotide and deduced amino acid sequence conservation between rabbits, humans, and mice. In situ hybridization indicated UBCi expression exclusively in the luminal epithelium of the endometrium while glandular epithelium, trophoblast, and myometrium were negative. Expression was specific for epithelial cells at implantation sites and was not detected in non-implant-site endometrium. UBCi mRNA was detected in both the mesometrial and antimesometrial epithelial cells of the implantation sites, sites undergoing both differentiation and/or apoptosis. These results identify a group of differentially expressed genes in the endometrium including UBCi and provide new focal targets for studying processes controlling cellular remodeling during implantation. The important roles of ubiquitination in controlling the activities and turnover of key signaling proteins suggest potential roles in controlling critical aspects of implantation.

implantation, uterus

INTRODUCTION

Blastocyst attachment and implantation in the uterine endometrium is delineated by complex differentiation steps, each requiring its own complement of newly induced genes. The acquisition of uterine receptivity is preceded by changes in both epithelial and stromal cells that provide for both embryo attachment and survival in the preimplantation Supported through NIH cooperative agreement U54 HD 37321 as part of the Specialized Cooperative Centers Program in Reproductive Research and by NIH grant AR 45943 to A.R.B. 2 Correspondence: Michael H. Melner, Department of Obstetrics & Gynecology, Vanderbilt University School of Medicine, B1100 Medical Center North, 1161 21st Ave. South, Nashville, TN 37232. FAX: 615 343 8881; e-mail: [email protected] 1

Received: 25 June 2003. First decision: 5 July 2003. Accepted: 14 October 2003. Q 2004 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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of immune-response genes and the increased expression of cell cycle- and keep-proliferation-related genes at implantation sites [13]. A recent study in the rat successfully employed suppressive subtractive hybridization to identify genes in the receptive endometrium induced by decidualization [14]. In the current studies, we screened for differentially expressed implantation-dependent endometrial genes in the rabbit, a model system allowing microdissection of the endometrium at implantation sites free from myometrium and blastocysts. The studies revealed the differential expression of several genes, including an uncharacterized ubiquitin-conjugating enzyme UBCi. This gene has the potential to play critical roles in the modification, targeting, and/or turnover of specific proteins during embryo implantation and the cellular remodeling accompanying endometrial differentiation. MATERIALS AND METHODS

Animals Estrous and timed pregnant New Zealand White rabbits [15] were obtained from Myrtle’s Rabbitry (Franklin, TN). The animals were housed and treated at the Vanderbilt University Medical Center Animal Care Center in accordance with National Institute of Health and U.S. Department of Agriculture standards. Animals were killed with sodium pentobarbital and organs were immediately removed. Uterine segments were either used for RNA extraction or embedded in OCT (Tissue-Tek, Sakura, Tokyo, Japan), frozen in liquid nitrogen, and stored at 2808C until used for cryosectioning.

Isolation of Differentially Expressed cDNAs Endometrium was microdissected from estrous uteri and from implant and interimplant regions of Day 6¾ and Day 8 pregnant rabbit uteri (samples representing three separate animals per group) and frozen at 2808C until used for RNA extraction. Poly(A)1 RNA was isolated from frozen tissue samples using an mRNA Isolation Kit (Stratagene, Inc., La Jolla, CA). The mRNAs were then used to prepare cDNA and to perform cDNA subtraction/suppression hybridization using the PCR-Select cDNA Subtraction Kit (Clontech, Inc., Palo Alto, CA) with modifications. Slight changes in the PCR amplification protocol (altered annealing temperatures and extended amplification cycles) were introduced to optimize the signal: noise ratio for these samples. Differentially expressed cDNAs were subcloned into pCR II-TOPO (Invitrogen Life Technologies, Inc., Carlsbad, CA). The Vanderbilt University DNA Sequencing Shared Resource performed sequencing of selected clones.

Northern Blot Analysis Isolation of total RNA from multiple tissues was performed using a RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). After extraction and column purification, RNA samples were quantified by absorbance at A260 and stored under excess ethanol at 2208C. Northern analysis was conducted using dimethyl sulfoxide-glyoxal RNA denaturation and agarose gel electrophoresis as described [16]. RNA samples (1–5 mg of poly(A)1 or 20 mg of total) or RNA size standards (Life Technologies, Inc., Rockville, MD) were denatured in 1.0 M deionized glyoxal, 50% (v/v) dimethyl sulfoxide, 10 mM sodium phosphate (pH 7.0) in a reaction volume of 25 ml for 1 h at 508C and resolved using agarose (1.5% w/v in 10 mM sodium phosphate, pH 7.0) gel electrophoresis with 10 mM sodium phosphate (pH 7.0). RNA was transferred to nylon membranes (Duralon-UV; Stratagene, Inc.) by capillary action with 203 SSC (13 SSC 5 0.15 M NaCl, 15 mM Na3 citrate), and fixed by ultraviolet (UV)-crosslinking and baking for 1 h at 808C. Glyoxal was removed from the immobilized RNA by incubation in 0.1 M sodium phosphate (pH 9.0) for 1 h at 658C. The blots were prehybridized in hybridization buffer (50% v/v deionized formamide, 53 SSC, 25 mM sodium phosphate buffer [pH 7.0], 5% w/v SDS, 0.1% w/v BSA, 0.1% w/v Ficoll 400, 0.1% w/v polyvinylpyrrolidone, and 0.05% w/v salmon sperm DNA) for 1 h at 428C prior to addition of the [a32P]dATP-labeled cDNA probe. The cDNA probes were generated using random primers and exo2 Klenow fragment (Prime-it II kit; Stratagene, Inc.). Probes were purified from unincorporated nucleotides using NucTrap columns (Stratagene, Inc.). After 18 h of hybridization, the blot was washed once with 23 SSC, 1% (w/v) SDS at 458C for 15 min; twice with

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23 SSC, 0.1% (w/v) SDS at 458C for 15 min; and once with 0.13 SSC, 0.1% (w/v) SDS for 10 min at 608C. Autoradiography was performed at 2708C with intensifying screens using Kodak BioMax MR film (Eastman Kodak, Rochester, NY). Following autoradiography, blots were stripped of probe by washing for 15 min at 908C in 10 mM sodium phosphate (pH 7.0), and rehybridized with a [a-32P]dATP-labeled probe for a constitutively expressed gene-encoding ribosomal protein S2 [17]. After hybridization, the blot was washed as described above. Autoradiographs were scanned using an Agfa Arcus II flatbed scanner (Agfa, Mortsel, Belgium) and saved as TIFF files.

Construction and Screening of cDNA Library Separate Day 6¾ and Day 8 pregnant rabbit endometrial unidirectional cDNA expression libraries were constructed in the ZAP express Lambda phage vector using a Lambda library construction kit (Stratagene, Inc.). The cDNA libraries were size selected on Sepharose CL-2B columns prior to ligation into the ZAP express vector. Following packaging, the libraries were titered, plated at 30 000 plaque forming units/plate, and then these unamplified libraries were screened with the initial differentially expressed cDNA inserts. Duplicate lifts from each of 12 plates were made with Duralon-UV filters. The filters were washed (1 min with 0.5 M NaOH/1.5 M NaCl, 3 min with 0.5 M Tris/1.5 M NaCl, 30 sec with 23 SSC), dried, and fixed by UV crosslinking. The filters were prehybridized in hybridization buffer and probed with a [a-32P]dATP-labeled cDNA probe as described above. The filters were then washed twice with 23 SSC, 0.1% (w/v) SDS at 458C for 15 min, and twice with 0.53 SSC, 0.1% (w/v) SDS for 15 min at 458C. Autoradiography was performed at 2708C overnight with intensifying screens using Kodak BioMax MR film. Positive plaques were cored and placed in chloroform in SM buffer (50 mM TrisHCl pH 7.5, 0.15 M NaCl, 10 mM MgSO4, 0.01% gelatin). Plaque purification was achieved by dilution and secondary and tertiary rounds of screening performed in a similar manner. Plasmids were excised from the phage using helper-phage-assisted excision. Sequencing of the cDNA clones was performed by the Vanderbilt University DNA Sequencing Shared Resource.

In Situ Hybridization Nonisotopic in situ hybridization was performed as described previously [10, 18]. Briefly, sense and antisense riboprobes were prepared using 1 mg of linearized plasmid in 20-ml transcription reactions containing SP6 or T7 polymerase; 13 transcription buffer; 20 U RNase inhibitor; 1 mM each of ATP, CTP, GTP; 0.65 mM UTP; and 0.35 mM Digoxigenin-UTP. Riboprobes were denatured for 5 min at 808C and diluted in hybridization buffer (50% formamide, 10% dextran sulfate, 43 SSC, 13 Denhardt reagent, 0.5 mg/ml heat-denatured herring sperm DNA, and 0.25 mg/ml yeast tRNA). Uterine cryosections were fixed with formaldehyde, treated with proteinase K in PBS, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer, pH 8.0, and hybridized overnight at 608C with digoxigen-labeled riboprobes. Slides were rinsed in 23 SSC, treated with RNase A, and subjected to stringency washes in 23 SSC, 50% formamide at 508C, and then in 13 SSC, followed by 0.53 SSC at room temperature. Hybridized riboprobes were detected using antidigoxigenin antibody conjugated to alkaline phosphatase and color development in 100 mM TrisHCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2 containing 0.34 mg/ml nitroblue tetrazolium, 0.17 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, 10 mM N-ethyl malemide and 1 mM levamisole to inhibit endogenous alkaline phosphatase.

Sequence Analysis The following uniform resource locators (URLs) were utilized for sequence analysis: http://www.ncbi.nlm.nih.gov/ [19, 20], http://www. expasy.ch/tools/scnpsit1.html, and http://www.ebi.ac.uk/swissprot/ [21]. Sequence alignment was performed using both ClustalW (http://www2. ebi.ac.uk/clustalw/) and Vector NTI (InforMax Inc., Frederick, MD).

RESULTS

Identification of Differentially Expressed Genes in the Endometrium at Implantation

A cDNA subtraction/suppression hybridization technique was employed to identify differentially expressed gene transcripts in implantation site endometrium from Day

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TABLE 1. Implantation-dependent cDNAs identified by cDNA subtraction/suppression hybridization. Previously identified implantation upregulated cDNAs

References

b-Haptoglobin Fibronectin (isolated twice) Galectin-3 Newly identified implantation upregulated cDNAs Ubiquitin-conjugating enzyme (UBCi) Ubiquitin-specific protease (Tre2 oncogene) Sphingomyelin delta-4 desaturase Hypothetical protein FLJ32122 (moderately similar to TRG) Hypothetical protein BM-009 Dynein intermediate chain-2 Channel kinase-1 PI-3-kinase-related kinase NCK-associated protein-1 (NAP-1) Acid ceramidase (N-acylsphingosine amidohydrolase) Catenin (cadherin-associated protein) Mitochondrial ribosomal protein S18 (nuclear encoded) Mitochondrial processing peptidase beta Malate dehydrogenase 1 TAF1 RNA polymerase II (TATA-binding protein-associated factor) NADH dehydrogenase

[22] [23–27] [28, 29] % Homology 100 93 91 89 93 93 93 95 93 89 84 87 92 91 93 99

Species* Rb Hu Hu Hu Hu Hu Hu Hu Hu Hu Hu Hu Hu Hu Hu Rb

GenBank Number AY330351 NM004505 AF466375 AK056684 NM016623 BT007130 AF346629 NM015092 NM013436 BC016436 NM001331 NM016067 AF054182 NM005917 NM138923 AJ001588

Unknown implantation upregulated cDNAs 9 different clones * Species are abbreviated: Hu, human; Rb, rabbit.

8 (implantation initiation) compared with Day 6¾ (embryo attachment) pregnant rabbits. Initially, 29 cDNAs were sequenced from this differential screen and are listed in Table 1. The cDNAs can be categorized in three groups: 1) cDNAs that have been previously reported in the literature to be upregulated in the endometrium during implantation, 2) cDNAs newly identified to be upregulated at implantation with functional implications based on their sequence homologies, and 3) unknown cDNAs that have no current homologies in the GenBank database. In the first category, we have identified multiple cDNAs identical to transcripts reported in the literature to be upregulated during implantation (Table 1). These include b-haptoglobin, previously shown to be strongly induced in epithelial cells of the rabbit endometrium during implantation [22]; fibronectin, shown to be upregulated in the primate endometrium during implantation [23–27]; and galectin-3, a galactoside-binding lectin [28, 29]. The identification of these clones in the limited pool of cDNAs examined to date represents a proof-of-principle that the differential gene expression cloning methodologies utilized were effective. A second distinct group of clones were also isolated that are newly identified as being upregulated at implantation in the uterus. These include two clones related to the ubiquitination pathway, a novel ubiquitin-conjugating enzyme, UBCi (i for implantation), and a new ubiquitin-specific protease/ Tre2 oncogene. These genes are novel in their association with implantation, their lack of characterization in published studies, and their absence from current commercial human and mouse microarray chips. UBCi was examined in further detail in these studies. Interestingly, multiple cDNAs encoding mitochondrial proteins were also identified in this limited sampling of differentially expressed genes. Cloning of Full-Length UBCi cDNA

One of the cDNAs identified in the cDNA subtraction/ suppression hybridization screen was a novel member of the ubiquitin-conjugating enzyme family. This cDNA was

selected for further characterization due to the importance of the ubiquitination pathway in regulating multiple critical signaling pathways. The cDNA fragment was used as a probe to screen a Day 8 pregnant endometrial cDNA library for full-length clones. A total of 18 independent cDNA clones were obtained and the consensus sequence of UBCi is shown in Figure 1. The cDNA was 2.9 kilobase (kb) in length with over 200 base pairs (bp) in 59 noncoding sequence and 1.4 kb in 39 noncoding sequence. The 39 noncoding sequence has two polyadenylation signal sequences (AAUAAA), one at 1800 bp and another at 2872. The relatively long 39 noncoding sequence also has four AUUUA sequences which are, in specific cases, involved in the targeting of mRNAs for rapid turnover and are present in the 39 noncoding regions of transcripts with short half-lives such as COX-2 and IL-8 [30, 31]. The deduced amino acid sequence encodes a 369 amino acid protein with remarkably high sequence identity with the deduced human and mouse sequences (95% and 94% sequence identity, respectively). The human ortholog in GenBank is entitled ‘‘similar to NICE-5’’. The acronym NICE-5 refers to newly identified cDNAs in epidermal differentiation complex [32]. While this title has been used in the sequence database, it does not accurately title the gene relating to its predicted function. The high sequence identity is underscored by the observation that the carboxyterminal 185 amino acids of the 369 residue protein containing the catalytic domain is 100% identical between rabbits, humans, and mice. The evolutionary conservation of sequence is further indicated by comparison of the last 100 amino acids of the rabbit, human, and mouse with the pufferfish (Fugu) as shown in Figure 2. This region is 100% identical in the three mammalian species and 97% identical in the pufferfish [33]. The deduced amino acid sequence homology was examined further to determine the similarity of the ubiquitinconjugating enzyme catalytic domain in the carboxy-terminal half of the protein to known E2 enzymes. Within this


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FIG. 1. Nucleotide and deduced amino acid sequence of UBCi. The double underlined sites indicate the two polyadenylation signal sequences. Bold underlines indicate the multiple AUUUA sequences described in the text.

FIG. 2. Comparison of amino acid sequences of UBCi orthologs from rabbit, human, mouse, and Fugu fish showing strong conservation of sequence. The three invariant residues of the predicted catalytic domain are highlighted by black dots above the line.

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FIG. 3. Northern blot probed for UBCi expression at the implantation sites of endometrium (left panel) and stripped/reprobed for a constitutive control gene, ribosomal protein S2 expression (right panel). Poly(A)1 RNA from Day 6¾ and Day 8 pregnant endometrium.

domain, there are three invariant residues as highlighted in Figure 2. First, there is a proline residue that precedes the catalytic site by 20 residues. This is followed by a cysteinyl residue, which is the active conjugation site for ubiquitin. Last, there is an invariant tryptophan separated from the cysteinyl residue by eight amino acids. The presence of these critical, invariant amino acid residues within the putative active site of UBCi, as well as the high degree of sequence homology in this region with other E2 enzymes, shows that UBCi has all the characteristic residues of a catalytically active ubiquitin-conjugating enzyme. Another interesting point concerning the amino acid sequence of UBCi is its length. The majority of active ubiquitin-conjugating enzymes of the E2 class are significantly shorter in length, ;200 residues versus 369 for UBCi. Although there are a few sequences in GenBank encoding putative E2 enzymes which are similar in length to UBCi, these proteins have not been characterized to date for their biochemical and enzymatic activities. One hypothesis concerning this extended amino terminal domain is that it may function in determining the specificity of the enzyme for its substrates, both cellular proteins and E3 ubiquitin ligase proteins, which interact with E2 during the terminal ubiquitin-conjugating step [34]. Differential Gene Expression

Northern blot analysis revealed the upregulation of UBCi mRNA during implantation (Fig. 3). Endometrial tissue microdissected from the site of implantation of Day 6¾ shows low-level expression while a significant increase (12-fold by scanning of the autoradiographs) is evident at Day 8. The UBCi mRNA ran as two transcripts of different sizes, primarily at 3.2 kb with low levels of a 2-kb transcript. Reprobing the blots with a cDNA probe for ribosomal protein

FIG. 4. Northern blot analysis of UBCi expression (top) and constitutive S2 expression (bottom) in different tissues. Lanes: 1, adrenal; 2, ovary; 3, prostate; 4, small intestine; 5, heart; 6, skeletal muscle; 7, brain; 8 liver.

S2, a constitutively expressed gene [35], confirmed loading of the lanes with equivalent levels of RNA (Fig. 3). The UBCi gene demonstrated a relatively high degree of tissue specificity. We examined a number of different tissues (Fig. 4) but observed expression in just the endometrium during implantation, the ovary, and the liver. The 3.2kb and 2-kb transcripts were both observed in all three of these tissues. UBCi mRNA Exhibits Endometrial Epithelial Cell-Specific Expression

The cells expressing UBCi were of interest due to the heterogeneous nature of endometrial tissue with epithelial cells, stromal cells, vascular cells, and immune cells as well as decidualizing stromal cells present. Nonisotopic in situ hybridization was performed using digoxigenin-labeled cRNA probes. As shown in Figure 5, Day 8 pregnant implantation site endometrium exhibited a strong signal for UBCi mRNA in luminal epithelial cells. The signal was absent from glandular epithelial cells, stromal cells, myometrial cells, and from trophoblast tissue, including the trophoblast knobs attached to the luminal surface of implantation sites. Sections across the entire implantation chamber indicated strong expression of UBCi in the placental folds of mesometrial epithelial cells as well as in antimesometrial epithelial cells (Fig. 6A). As a negative control, sections hybridized with a sense cRNA for UBCi were negative for any signal (Fig. 6B). Expression of UBCi RNA was undetectable in the Day 8 pregnant non-implant-site endometrial tissue, indicating the specificity of expression for the sites of implantation (Figure 6, Panel C). Endometrial


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FIG. 5. Nonisotopic in situ hybridization showing UBCi mRNA expression in the antimesometrial luminal epithelium (le) of a Day 8 implantation site. Note the absence of detectable UBCi expression in the trophoblast (t), glandular epithelium (ge), or myometrium (m). Magnification 3300.

cells from Day 6¾ pregnant uteri just prior to implantation were also largely negative for signal. No expression was detected in the embryo (em) or stroma (st) shown in Figure 6E. These data suggest that UBCi expression is specific for endometrial luminal epithelial cells at the sites of implantation during the critical initiation of this process.

DISCUSSION

The success of the differential screening methodologies, as evidenced by the identification of multiple known implantation-induced genes as well as novel uncharacterized genes, has great potential for further mining of differenFIG. 6. Nonisotopic in situ hybridization of Day 8 (A, B, D, and E) and Day 6¾ (C) implantation chambers hybridized with antisense (A, C, D, E) or sense (B) digoxigen-conjugated riboprobes to UBCi. At Day 8, strong UBCi expression is detected in the endometrial luminal epithelium (en) (A, D, E) but not the myometrium (m). Staining is detected in the luminal epithelium of the placental folds (pf in A), which are shown in high magnification in D. The antimesometrial luminal epithelium (ep) also displays high UBCi expression but no expression is detected in the embryo (em) or stroma (st) (E). Day 8 controls probed with sense probes display no hybridization (B). No expression of UBCi is detected at Day 6¾ (C).

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tially expressed genes. A number of mitochondrial genes were identified in the differential screening protocol. The significance of this is not known at this time but alterations in mitochondrial function are integral to pathways of both differentiation and, alternatively, apoptosis. This finding indicates that examinations of mitochondrial changes may be of merit to understanding important keys to early implantation events. Our studies identify UBCi, a new member of the E2 ubiquitin conjugation enzyme family, which is dramatically upregulated in endometrial epithelial cells at the sites of embryo implantation. The remarkable conservation of UBCi sequence homology between rabbits, mice, and humans at the nucleic acid and deduced amino acid levels, particularly in the predicted catalytic domain of the carboxyterminal half of the protein, suggests a putative catalytic function for this gene. The amino terminal extended domain of UBCi is of interest because this domain is the predicted substrate recognition domain of ubiquitin-conjugating enzymes. Multiple enzymes are involved to effect ubiquitination: the E1 enzyme activates ubiquitin and transfers it to a member of the E2 class of ubiquitin-conjugating enzymes; the E2 enzymes couple either directly with protein substrates or with E3 ubiquitin ligases and transfer ubiquitin to these substrates. There are a relatively large number of different E2 and E3 enzymes that display specificity for groups of specific substrates [36]. Monoubiquitination and polyubiquitination have been shown to target proteins for degradation in the 26S proteosome [37], to target proteins to the endocytic pathway, and also to functionally activate specific proteins [38, 39]. Increasing evidence indicates a role for ubiquitination in the regulation of critical signal transduction pathways, in some cases by degradation of critical signaling molecules, but in others by enhancing their activity. Proteins that are affected include transcription factors such as the steroid receptors (estrogen, progesterone, glucocorticoid, and androgen receptors) [40–43], apoptosis proteins [44–46], cell cycle regulatory proteins [47], and tumor suppressors [48]. An example of a ubiquitin-mediated protein activation is TRAF6, a signal transducer in the NFkB pathway, which activates IkB kinase. TRAF6 is a ubiquitin ligase (E3 protein) that functions with the ubiquitin-conjugating enzyme Ubc13 to catalyze ubiquitination and resulting activation of IkB kinase [39]. This step activates IkB kinase but is not involved in targeting proteins for degradation in the proteosome because proteosome inhibitors do not alter this pathway. In the apoptotic pathway, proteins called IAPs (inhibitors of apoptosis) oppose caspases and thus act to prevent apoptosis [46]. These IAP proteins have ubiquitin ligase activity, and proapoptotic proteins such as Reaper can stimulate IAP-mediated autoubiquitination, leading to loss of IAPs and increased apoptosis [46]. These are examples of critical signal transduction control points that are regulated by ubquitination. The pathways affected by UBCi are currently unknown. It is interesting that UBCi is upregulated in epithelial cell populations of the endometrium that presumably undergo apoptosis and eventual sloughing (i.e., the antimesometrial luminal epithelial cells) as well as in the cell populations that undergo dramatic differentiation during implantation (i.e., mesometrial epithelial cells). The potential role of UBCi in these processes will require careful molecular dissection of the specific protein substrates of this enzyme and the resulting changes in the function and/or levels of these protein substrates during implantation. Previous studies

have also noted increased protein ubiquitination as well as specific ubiquitinated proteins in the endometrium during the implantation window [49–55]. Of particular note, Pru et al. [56] have found the upregulation of members of the 1-8 family of proteins, putative E2 conjugating enzymes, in the pregnant bovine uterus. The expression pattern of the 1-8 protein mRNAs appears to differ from UBCi in that UBCi is expressed predominantly in the luminal epithelial cells and is undetectable in the glandular epithelial cells and stromal cells. In contrast, the 1-8 protein mRNAs were heavily localized to glandular epithelium with lesser expression in luminal epithelium and stroma [56]. While these patterns of expression differ, the temporal expression of multiple E2 conjugating enzymes in different uterine cell populations during implantation and early pregnancy suggests important roles for the ubiquitination pathway. The potential involvement of UBCi in these processes is of clear interest given the correlation between the predicted enzymatic activity and the observed results. It is also of interest that some key epithelial signaling proteins and proteins involved in cytoskeletal reorganization undergo dramatic changes during implantation. For example, there is a dramatic loss in detectable progesterone receptors in the luminal epithelial cells during implantation in multiple species [57, 58]. In addition, there is a large down-regulation in cytokeratin 18 involved in the cytoskeletal reorganization of these cells during this process [9]. The mechanisms for the rapid and specific loss of these key proteins at this time are not currently known. It is of interest to test whether specific ubiquitination by upregulated members of the ubiquitination pathway such as UBCi are involved in this process. Our identification of a ubiquitin-specific protease/Tre2 oncogene differentially expressed in the endometrium at implantation (Table 1) is interesting in light of recent evidence that this gene is absent in most mammals with the exception of primates and rabbits [59]. This gene is an apparent fusion during evolution of two genes, the USP32 gene and TBC1D3 gene [59], and was originally isolated by transfection screens for genes that transform NIH 3T3 cells [60, 61]. Previous data have indicated that expression of this gene is fairly specific for the testis. The high species specificity of the existence of this gene may make the rabbit a critical model for implantation-based studies of its functions. Members of the E2 class of ubiquitin-conjugating enzymes function to transfer activated ubiquitin from the E1 enzyme, either directly to specific cellular protein substrates or coupled with E3 ubiquitin ligases to these substrates [62]. There are estimated to be 49 different E2 or E2-like ubiquitin-conjugating enzyme genes and an estimated 600 E3 ubiquitin ligase genes. This large number of proteins with similar catalytic activity suggests the individual proteins have distinct specificities for particular cellular substrates [62]. The specificity of these enzymes has also been substantiated by naturally occurring mutations and/or targeted gene disruption studies, which suggest specific, nonlethal phenotypes by multiple ubiquitin-conjugating enzyme genes [63]. Therefore, dramatic upregulation of the UBCi gene initiated by implantation is a unique system for investigating the specific modifications and substrates of this enzyme and the resulting cellular changes initiated by its activity. ACKNOWLEDGMENT The authors wish to thank Michael S. Melner for critical comments on the writing of the manuscript.


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