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Regular Article |
a Departments of Physiology and Obstetrics and Gynaecology, The University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, implantation, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uterine receptivity, or maximal sensitization for the decidual cell reaction, in the rodent is maternally regulated by estrogen and progesterone [3, 4]. In rats, a minimum of 48 h of progesterone exposure induces a prereceptive neutral state when the uterus exhibits suboptimal sensitization for the decidual cell reaction. Estrogen exposure following progesterone priming (in vivo referred to as the nidatory estrogen surge) induces a state of refractoriness within 36 h, an endometrial environment that can neither support blastocyst survival nor respond to a deciduogenic stimulus [5]. A short phase of receptivity/maximal sensitization is a transient event between the neutral and refractory states. Although it is clear that estrogen and progesterone regulate the events that lead to uterine receptivity/maximal sensitization for the decidual cell reaction, little is known of what mediates the hormone-induced onset of this phenomenon.
Many locally produced molecules have been implicated as having a role in implantation, including members of the epidermal growth factor (EGF) family (EGF, heparin-binding EGF, and amphiregulin) [6–8], the EGF receptor [9], calcitonin [10], colony-stimulating factor (CSF-1) [11], interleukin-1 (IL-1) and its receptor [12], cyclooxygenase-2 (COX-2) [13], and leukemia inhibitory factor (LIF) [14]. Of these, only LIF has been shown to act specifically at the onset of the receptive/maximally sensitized phase [14, 15]. Despite a growing list of molecules known to be involved in the implantation process, and specifically genes associated with the onset of uterine receptivity, the specific cascade of molecular events that direct the steroid hormone-mediated onset of uterine receptivity remains to be determined.
To identify genes not previously associated with the onset of uterine receptivity, we utilized the technique of suppression subtractive hybridization (SSH) [16]. Briefly, to enrich for genes preferentially expressed at the time of maximal sensitization for the decidual cell reaction, mRNA from rat uteri on the equivalent of Day 4 of pseudopregnancy (neutral) was subtracted from mRNA isolated from uteri on the equivalent of Day 5 of pseudopregnancy (sensitized). Herein we report the identification, cloning, and partial characterization of a novel gene, which we have termed uterine sensitization-associated gene-1 (USAG-1), expressed within the rat endometrium at the time of maximal sensitization/uterine receptivity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Sprague-Dawley rats (200–225 g body mass; Harlan Sprague-Dawley, Indianapolis, IN) were housed in temperature- and light-controlled conditions (14L:10D with lights-on from 0500 h to 1900 h) with free access to food and water. Animals were ovariectomized under ether anesthesia (BDH, Toronto, ON, Canada) and allowed at least 5 days recovery. To obtain rats with uteri differentially sensitized for the decidual cell reaction, estradiol (E2) and progesterone (P4) (Sigma Chemical, St. Louis, MO) were administered subcutaneously (s.c.) in sesame oil as previously described [17]. Some rats received a bilateral injection of sesame oil into the uterine lumen on the equivalent of Day 5 of pseudopregnancy to induce decidualization [18]. Rats receiving intraluminal injections of sesame oil on Day 5 and their controls continued to receive daily s.c. injections of E2 and P4 in sesame oil until they were killed [17]. Rats were killed by decapitation on the morning of each day of the equivalent of Days 1–10 of pseudopregnancy and uteri were collected. For experiments involving the induction of pregnancy, virgin female rats at proestrus were placed with males; the presence of sperm in the vagina on the following day was marked as Day 1 of pregnancy. Uteri were collected on Days 4, 5, and 6 of pregnancy. Day 6 pregnant animals received an i.v. injection of 0.5% Evans blue 15 min prior to killing to visualize the implantation sites [19], thereby allowing implantation sites to be separated from interimplantation sites. All procedures involving animals were performed in accordance with the guidelines of the Canadian Council on Animal Care and The University Council on Animal Care at the University of Western Ontario.
Subtraction Hybridization
Uteri from rats at the equivalent of Day 4 or Day 5 of pseudopregnancy were obtained using a previously described protocol [17]. The mRNA from whole uteri was isolated using a QuickPrep mRNA purification kit (Amersham Pharmacia Biotech, Baie d'Urfé, QC, Canada). Subtraction hybridization was carried out using a Clontech PCR-Select cDNA Subtraction Kit (Clontech Laboratories, Palo Alto, CA) as described by the manufacturer. Briefly, mRNA from Day 4 uteri was used to make driver cDNA and mRNA from Day 5 uteri was used to make tester cDNA. The subtraction was carried out so that a pool of cDNAs enriched for sequences expressed preferentially in Day 5 uteri was generated. A subtracted cDNA library was made by cloning the cDNAs generated in the subtraction hybridization directly into the pGEM-T easy vector (Promega, Madison, WI). Clones containing subtracted cDNAs were isolated by blue/white screening and their differential expression was confirmed by Northern blot analysis.
Northern Blot Analysis
Northern blot analysis was performed as previously described [20]. Briefly, total RNA from whole uteri or from isolated endometrium [21] was subjected to electrophoresis in a denaturing gel. The RNA was then transferred and cross-linked to Hybond-N membranes (Amersham Pharmacia Biotech) and hybridized at 60°C in Church buffer [22] in the presence of 32P-labeled DNA probes. Membranes were subsequently washed three times in wash buffer at 65°C and exposed to Kodak Biomax MS film (Eastman Kodak, Rochester, NY) at -70°C. 18S ribosomal RNA signal was used to determine the relative amounts of RNA loaded and transferred to the membrane [23].
Full-Length Cloning of cDNA
3' and 5' rapid amplification of cDNA ends (RACE), using the SMART RACE cDNA Amplification Kit (Clontech Laboratories), was used to obtain the full sequence information for USAG-1. The sequencing was done using an automated fluorescent ABI377 DNA sequencer (The John P. Robarts Research Institute DNA Sequencing Facility, London, ON, Canada). Searches for homology to Genbank database sequences, potential structural motifs, and open reading frame determination were done using the databases available on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) and the Prosite database of protein families and domains (http://ca.expasy.org/prosite/).
In Situ Hybridization
In Situ hybridization was performed as previously described [20]. Uterine horns from different stages of pseudopregnancy were collected and fixed in 4% paraformaldehyde for 24 h, rinsed in 2 changes of PBS, and processed for paraffin embedding. Sections (6 µm) were hybridized with digoxigenin (DIG)-labeled probes in a 50% formamide buffer at 55°C for 16 h. After hybridization, sections were treated with RNase A (20 µg/ml) at 37°C for 20 min and the DIG-labeled cRNA probe was detected using anti-DIG antibodies at a dilution of 1:500 (Roche Molecular Biochemicals, Laval, QC, Canada).
Statistical Analysis
Experiments quantifying changes in mRNA levels by Northern blot analysis were performed three times on separate groups of ovariectomized, hormone-treated rats (except when stated otherwise). The data were analyzed by within-blocks ANOVA, with experiments being considered blocks. Duncans multiple range test was performed to determine differences between groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By SSH, a pool of cDNAs, enriched in Day 5 specific mRNA, was generated, cloned into the pGEM-T easy vector, and individual clones identified by blue/white screening. Herein we report the identification of a novel gene, designated USAG-1, that is differentially expressed within the rat uterus during the peri-implantation period as indicated by Northern blot analysis (Fig. 1).
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DNA Sequencing and Sequence Homology Search
The full-length mRNA sequence for USAG-1 was obtained by 3' and 5' RACE. The resulting cDNA sequence was 1718 bases in length (Fig. 2; Genbank accession number AF411056). A search of nucleotide databases NCBI-nr, -est, -month [24] revealed 96% homology to several full-length, but uncharacterized, mouse cDNAs (Genbank accession numbers NM_025312, AK007935, AK007893, AK002396, AK007967, AK002240), 91% homology (over the open reading frame) to a partially sequenced human cDNA (clone DKFZp564D206, Genbank accession number AL050024), and high homology to a large number of expressed sequence tags (ESTs) from human, rat, mouse, and bovine.
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The USAG-1 cDNA contains a putative open reading frame of 621 base pairs (bp) (from bases 76–696), predicting a protein of 206 amino acids (Fig. 2). Searches for conserved protein domains demonstrated the presence of a possible C-terminal cystine knotlike motif (CTCK) from amino acids 75–170. Also, several potential N-glycosylation sites, N-myristoylation sites, protein kinase C, cAMP- and cGMP-dependent protein kinase, and casein kinase II phosphorylation sites were detected. Upon analysis of the putative amino acid sequence by the Target P program [25], a predicted secretion signal was identified in the first 23 amino acids of the N-terminus (Fig. 2). A sequence homology search of the putative protein sequence [24] revealed 98% sequence identity with the predicted amino acid sequence for the mouse cDNA 0610006G05 (Genbank accession number NM_025312) and 97% sequence identity with the putative protein encoded by the human cDNA clone DKFZp564D206. Both these protein sequences are predicted from uncharacterized cDNAs within the Genbank databases. Lower sequence homologies (38–40%) were observed with the protein sclerostin from rat, mouse, human, nonhuman primate, and bovine (Genbank accession numbers NP085073, NP077769, XP15444, AAK13457, AAK13453, respectively). Sclerostin is a recently identified secreted protein whose deletion is linked to sclerosteosis, a craniotubular bone remodeling disorder [26, 27]. Much of the homology centers around a cystine-rich region that is essential for the formation of the cystine knot. The conservation of the specific pattern of nine amino acids, eight cystines and a glycine, is consistent with USAG-1 being in a class of secreted proteins that contain the cystine knotlike motif; these include the transforming growth factor-ß superfamily, the Norrie disease protein, the mucins, and von Willebrand factor [26, 27].
Temporal Pattern of USAG-1 mRNA Expression Within the Pseudopregnant Rat Uterus
Northern blot analysis on differentially sensitized uteri was used to assess whether USAG-1 mRNA induction was restricted to the maximally sensitized Day 5 uteri (Day 5 inter E2). Briefly, animals were ovariectomized and treated with a hormone protocol that differentially sensitizes the endometrium for the decidual cell reaction [17]. Total RNA was isolated from endometrium from animals that were at the equivalent of Day 4, Day 5, or Day 6 of pseudopregnancy as well as from two groups that were temporally correct but hormonally nonsensitized (Day 5 high E2 and Day 5 low E2). As shown in Figure 3, USAG-1 mRNA was expressed at low levels on Day 4, was dramatically upregulated on Day 5, at the time of maximal sensitization for the decidual cell reaction, and was again at low to undetectable levels within the Day 6 refractory uterus. Day 5 uteri from animals that did not receive E2 on the evening of Day 4 (Day 5, low E2) also showed low levels of USAG-1 mRNA expression. However, animals that received a dose of E2 on the evening of Day 4 that is too high to sensitize the endometrium for the decidual cell reaction (Day 5, high E2) showed USAG-1 mRNA expression at levels comparable with the maximally sensitized uteri. To further examine the expression of USAG-1 mRNA throughout pseudopregnancy, Northern blot analysis on total endometrial RNA from Days 1–10 of pseudopregnancy, in the presence and absence of decidualization, was performed. As seen in Figure 4, USAG-1 mRNA induction is restricted to the Day 5 sensitized endometrium. Some animals received an intrauterine injection of sesame oil at around noon on Day 5 to initiate decidualization (stimulated) while others did not (nonstimulated). USAG-1 mRNA levels were not maintained after Day 5 regardless of the presence or absence of decidualization.
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Localization of USAG-1 mRNA Expression in Differentially Sensitized Rat Uteri
In situ hybridization experiments using DIG-labeled cRNA probes specific for USAG-1 verified that an induction of USAG-1 mRNA occurs in the uteri of Day 5 intermediate E2 and Day 5 high E2 animals but not in the uteri of Day 4, Day 6, or Day 5 low E2 animals. This upregulation appears to be restricted to the uterine glandular epithelium (Fig. 5). There is faint expression in the surrounding stroma and possibly the luminal epithelium that is above background, but this does not appear to change with respect to the treatment group.
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Hormonal Control of USAG-1 mRNA Expression in the Pseudopregnant Uterus
To examine the hormonal control of USAG-1 mRNA expression within the uterus in vivo, animals were ovariectomized and injected s.c. with a single dose of either 1.0 µg of E2, 4 mg P4, 1.0 µg E2 + 4 mg P4, or vehicle (sesame oil). Uteri were collected 18 h after injection and total RNA was isolated for Northern blot analysis (Fig. 6). As indicated by ANOVA of log-transformed densitometric data, both E2 and P4 significantly (P < 0.01) increased USAG-1 mRNA levels, by approximately sixfold, and their combined effect was additive on a geometric scale. E2 and P4 in combination resulted in mRNA expression comparable with that in the Day 4 pseudopregnant uterus. A large increase in mRNA levels for USAG-1 was seen only in the Day 5 intermediate E2 uterus.
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Time Course of USAG-1 mRNA Induction
To determine the time-course of USAG-1 mRNA induction following E2 exposure, we treated ovariectomized rats with the same hormone protocol used previously to maximally sensitize the endometrium. Total uterine RNA was collected 0 and 30 min and 1, 2, 6, 12, and 18 h after E2 administration on the afternoon of Day 4. Time zero corresponds to a Day 4 neutral endometrium, and the 18 h time-point corresponds to the time of maximal sensitization on the equivalent of Day 5. Uterine USAG-1 mRNA transcripts were only faintly detected at 2 h (confirmed with longer exposure—not shown) but were 10-fold higher by 6 h (Fig. 7). USAG-1 mRNA induction reached a maximum 12 h after E2 administration (approximately 70-fold increase from 2 h) and was maintained at this level through to 18 h. Previous Northern blot analysis indicated that USAG-1 mRNA levels on the equivalent of Day 6 of pseudopregnancy (24 h after the 18-h time point in this experiment) are the same as those on Day 4 (Figs. 3 and 4).
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USAG-1 mRNA Expression During Pregnancy and Onset of Sensitization
To determine if similar changes in expression occur during pregnancy as observed in ovariectomized, steroid-treated animals, virgin rats were mated and uteri were collected on Day 4, Day 5, and Day 6 of pregnancy. USAG-1 mRNA expression during pregnancy has the same pattern of expression as in the ovariectomized hormone-treated rat, with low expression on Day 4, a rapid and large upregulation on Day 5, and a return to low levels by Day 6 in both implantation sites and interimplantation sites (Fig. 8).
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Further investigation of USAG-1 mRNA expression at the time of uterine sensitization was done using a model of delayed implantation. Ovariectomized rats were put on the same hormone protocol used to obtain sensitized uteri, but instead of administering E2 after 48 h of P4, P4 treatment was extended an additional 48 h before E2 was given. The expression of USAG-1 mRNA remained low within the uteri of rats in the absence of E2 administration. With E2 administration, USAG-1 mRNA was induced to levels similar to those in the pregnant or pseudopregnant Day 5 uterus (Fig. 8).
Tissue Distribution of USAG-1 mRNA Expression
We isolated total RNA from several tissues and investigated USAG-1 mRNA expression using Northern blot analysis. As seen in Figure 9, a low level of expression was detected in brain, kidney, and the female reproductive tract (cervix, vagina, uterus, oviduct, and ovary; all collected without regard to stage of estrous cycle). There was no detectable signal in the placenta from Days 12–20 of pregnancy. These sites of expression correspond to sources for ESTs that match the USAG-1 mRNA sequence in the databases. The expression level of USAG-1 mRNA in these tissues is substantially lower than that seen in the Day 4 neutral endometrium, which is, in turn, much lower than seen in Day 5 sensitized endometrium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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USAG-1 mRNA displayed a remarkable expression pattern within the rat endometrium during the peri-implantation period. Northern blot analysis showed very low mRNA levels within the neutral or Day 4 pregnant/pseudopregnant uterus. A dramatic upregulation was seen by Day 5, when the endometrium is maximally sensitized for the decidual cell reaction, but this increase was not maintained, as a return to previous levels was observed within 24 h, or by Day 6, of pregnancy or pseudopregnancy (Figs. 3, 4, and 8). The application of a deciduogenic stimulus on Day 5 did not affect the decline of USAG-1 mRNA following Day 5 (Figs. 4 and 8). Animals that did not receive E2 following 48 h of P4 priming (Day 5 low E2 group) did not have an increase in uterine USAG-1 mRNA expression, suggesting that E2 is required for induction. Supporting this was the observation that USAG-1 mRNA induction was absent in a model of delayed implantation until E2 was administered (Fig. 8). Further analysis of the hormonal regulation of this gene in vivo revealed that, while E2 and P4 had an additive effect on USAG-1 mRNA expression (geometric scale), a single dose of E2 and P4 in combination did not fully induce USAG-1 mRNA. A large increase in mRNA levels was seen only after a minimum of 48 h of P4 priming followed by administration of E2, the conditions required for the onset of uterine receptivity (Fig. 6). However, USAG-1 mRNA was also upregulated in the Day 5 high E2 group, animals temporally correct for uterine sensitization but hormonally nonsensitized. The concentration of E2 required to transform a P4 primed (neutral) endometrium to a receptive endometrium is critical because too high a dose does not result in maximal sensitization [28]. The Day 5 high E2 group received a dose of E2, following 48 h of P4 priming, that was sufficient to inhibit the onset of a maximally sensitized phase [17, 20].
These observations suggest that E2 exposure, only after at least 48 h of P4 priming, is required to significantly increase USAG-1 mRNA expression (Fig. 6). Also, the concentration of E2 required after the P4 priming does not appear to be as critical to the induction of USAG-1 mRNA as it is for the onset of uterine receptivity/maximal sensitization (Fig. 3). Taken together, it is unlikely that USAG-1 is involved in triggering the onset of uterine receptivity, but more likely, USAG-1 is involved in downstream events after the molecular cascade initiating the onset of receptivity has been started.
Northern blot analysis of various tissues showed that USAG-1 mRNA is expressed at low levels within the brain, kidney, and reproductive tract (Fig. 9). However, to date, the greatest level of expression identified (many fold higher than in any other tissue) is within the maximally sensitized endometrium. In situ hybridization experiments revealed that the glandular epithelium is the major site of USAG-1 mRNA expression (Fig. 5). The stroma, luminal epithelium, or myometrium did not appear to significantly express USAG-1 mRNA or demonstrate a change in expression levels over time. It has been well documented that an increase in protein synthesis and secretion from the glands occurs after the initiation of implantation and is maintained throughout early pregnancy [29]. However, evidence supporting a secretory event arising from the uterine glands that functions specifically in the onset of uterine receptivity is mounting. It has recently been reported that an increase in GRP 78 and Rab 11, molecules involved in the biosynthesis and secretion of proteins, occurs in the uterine glands around the time of maximal sensitization for the decidual cell reaction but is not sustained throughout early pregnancy [20, 30]. Also LIF, a molecule known to be required for the onset of uterine receptivity, is secreted from the uterine glands in mice primarily at the time of uterine receptivity [31]. Recently it has been shown that LIF expression is required only at the onset of receptivity, for the acquisition of the receptive state, and not for pre- or postimplantation embryogenesis or the maintenance of pregnancy [15]. It is thought that LIF interacts with its receptor expressed within the luminal epithelium to initiate the first steps in the onset of the uterine receptivity [31]. Although at present it is not known if USAG-1 mRNA is translated, a possible secretion signal has been identified in the first 23 amino acids of the putative protein sequence, indicating that it is also likely secreted by the glandular epithelial cells. USAG-1's function at the time of uterine receptivity remains unclear, but if secreted, it could have actions on the luminal epithelium or stroma to participate in the acquisition of the receptive phase (probably downstream of LIF) or it could act on the embryo itself.
In summary, USAG-1 mRNA has an extraordinary expression pattern within the receptive or maximally sensitized endometrium, with such intense induction for only a 24-h period. We are unaware of any gene investigated to date that has expression so tightly correlated with the receptive phase of uterine development. USAG-1 is an accurate and dramatic marker for uterine receptivity in the rat.
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FOOTNOTES |
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2 Correspondence: Thomas G. Kennedy, Dental Sciences Building, Room 2002, Departments of Physiology and Obstetrics and Gynaecology, The University of Western Ontario, London, ON, Canada N6A 5C1. FAX: 519 661 3827; tom.kennedy{at}fmd.uwo.ca
Received: 24 April 2002.
First decision: 10 May 2002.
Accepted: 6 June 2002.
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REFERENCES |
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