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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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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uterine receptivity corresponds to optimal sensitization for the decidual cell reaction [2], and the hormonal requirements for its onset in the rodent have been worked out in detail [1]. Exposure to progesterone for 48 h induces a pre-receptive "neutral" state when the uterus exhibits a suboptimal sensitization for the decidual cell reaction as well as conditions that allow for blastocyst survival, albeit in dormancy [3–5]. Exposure to estrogen can then induce a state of refractoriness within 36 h. A short phase of receptivity/sensitization appears as a transient event midway between the neutral and refractory states. In the nonreceptive or refractory state the endometrium is unable to respond to a deciduogenic stimulus, and the uterine environment becomes hostile to blastocysts [5]. Thus a model has been developed for the requirement of a time-dependent progesterone-estrogen sequence that must be followed in order to induce an endometrial receptive/sensitized state. The concentration of estrogen is also a critical component, as too much or too little will not produce a receptive endometrium [6, 7].
We exploited the tight hormonal requirements for the generation of a sensitized state and the technique of mRNA differential display reverse transcription-polymerase chain reaction (ddRT-PCR) [8] to identify gene expression that was unique to the sensitized endometrium. Briefly, ovariectomized rats were treated with progesterone (P4) and estradiol (E2) to generate animals differentially sensitized for the decidual cell reaction, and endometrial RNA from different stages of sensitization were compared using ddRT-PCR. PCR products (amplicons) that showed sensitization-specific expression patterns were isolated, identified, and characterized.
One such amplicon was identified as glucose-regulated protein 78 (GRP78). GRP78, also known as immunoglobulin heavy chain-binding protein, is a member of the heat shock 70 class of proteins [9]. It is a major calcium (Ca2+)-binding protein residing in the endoplasmic reticulum (ER) [10] where it functions as a molecular chaperon [11] and possibly in the translocation of nascent proteins across the ER membrane [12]. It is believed that GRP78 not only acts as a molecular chaperon, assisting in the proper folding of newly synthesized proteins, but also acts, through an unknown mechanism, to protect cells against a variety of physiological stresses [11]. GRP78 can be induced in response to a multitude of stimuli including glucose or oxygen deprivation, perturbation of intracellular Ca2+ stores, and the interruption of proper protein folding, transport, or processing (reviewed in [11]). In the present study we demonstrate the induction of GRP78 within the rat endometrium at the time of optimal uterine sensitization and characterize its temporal and spatial expression during the periimplantation period.
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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 under light- and temperature-controlled conditions (14L:10D; 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 4 days to recover. To obtain rats with uteri differentially sensitized for the decidual cell reaction, E2 and P4 (Sigma Chemical Co., St. Louis, MO) were administered s.c. in sesame oil as described previously [7]. Some rats received a bilateral injection of 0.1 ml of sesame oil into the uterine lumen on the equivalent of Day 5 of pseudopregnancy in order to induce decidualization [13]. After stimulation, these rats, and also controls that had not been stimulated, continued to receive 0.1 µg E2/4 mg P4 s.c. in sesame oil daily until they were killed [7]. Rats were killed by decapitation on the morning of each day of equivalent pseudopregnancy from Days 1 through 10. The uterine horns were isolated and cleaned in ice-cold saline, and the endometrium was separated from the myometrium as previously described [14]. 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.
Differential Display
Changes in gene expression during the periimplantation period were analyzed using the method of ddRT-PCR as described by Liang and Pardee [8] with some modifications. Endometrial samples were immediately homogenized in guanidinium isothiocyanate buffer, and total RNA was extracted using a single-step guanidine thiocyanate procedure [15]. Total RNA was treated with DNase (Gibco-BRL, Burlington, ON, Canada) and its integrity checked by gel electrophoresis. After DNase treatment the RNA was quantified by absorbance at 260 nm.
RT reactions were carried out using the 3' primer T11MC, where M was A, C, G, or T. The RT reactions contained 100 ng of RNA, 2.5 µM 3' primer (T11MC), 10 U of RNase inhibitor (Gibco-BRL), single-strength first strand buffer (Gibco-BRL), 20 µM dNTPs, and 200 U reverse transcriptase (Gibco-BRL) in a total volume of 20 µl. The reactions were carried out at 42°C for 90 min followed by 95°C for 5 min. Complementary DNA was then stored at -20°C until the PCR were done.
PCR reactions were performed using the same 3' primer as for the RT and one of 26 random 10-mer 5' primers as described by Bauer et al. [16], in this case 5'-GGTACTAAGG-3'. Each reaction was done in triplicate and contained single-strength PCR buffer (Gibco-BRL), 2.5 µM MgCl2, 2 µM dNTPs, 2.5 µM 3' primer, 0.5 µM 5' primer, 2.5 U of Taq polymerase (Gibco-BRL), 10 µCi of [
-35S]dATP (Amersham, Oakville, ON, Canada), and 2 µl of RT product. The final volume was 20 µl. Amplification was done for 40 cycles of 94°C for 30 sec, 42°C for 1 min, and 72°C for 30 sec, followed by extension at 72°C for 10 min. Five microliters of each PCR reaction was mixed with 2 µl of loading buffer (90% formamide/dye solution) and heated at 95°C for 10 min, then placed on ice. Samples were spun down and loaded on a standard 0.4-mm, 6% polyacrylamide-urea sequencing gel and run for 4 h at 50°C. Gels were transferred to filter paper, dried on a gel dryer (Bio-Rad Laboratories, Mississauga, ON, Canada), and exposed to Kodak Biomax MR x-ray film (Eastman Kodak, Rochester, NY) overnight.
Recovery and Reamplification of cDNA Amplicons
After the x-ray film was developed, cDNA bands of interest were cut out of the dried gel through the film and rehydrated in 100 µl of 10 µM Tris, 10 µM EDTA, pH 8.0 (TE); they were then boiled in a tightly capped microfuge tube for 15 min. Complementary DNA was recovered by ethanol precipitation in the presence of 0.3 M sodium acetate and 5 µl of glycogen (10 mg/ml; Sigma). The pellet was washed with 50 µl of 80% ethanol and resuspended in 10 µl of H2O. Five microliters of eluted cDNA was reamplified in a 40-µl volume using the same PCR conditions as for the differential display except that the dNTP concentration was 20 µM, not 2 µM, and no [-35S]dATP was added to the reaction. Twenty microliters of the reamplification reaction was run on a 1.5% agarose gel and stained with ethidium bromide for visualization. The remaining 20 µl was kept at -20°C for cloning.
Cloning and Sequencing of cDNA Amplicons
Reamplified cDNA amplicons were cloned into the pGEM T-easy vector (Promega, Madison, WI) using the provided TA cloning kit and following the manufacturer's instructions. Sequencing was done using an automated fluorescent ABI377 DNA sequencer (The John P. Robarts Research Institute DNA Sequencing Facility, London, ON, Canada).
Northern Blot Analysis
Ten micrograms of total endometrial RNA was denatured and subjected to electrophoresis in a denaturing gel as previously described [17]. RNA was then transferred to Hybond-N membrane (Amersham) by capillary transfer and cross-linked by exposure to 1.2 x 105 µJ/cm2 of UV energy (Hoefer Pharmacia Biotech, San Francisco, CA). Northern blot analysis was performed as described by Church and Gilbert [18], with some modifications. Briefly, membranes were prehybridized in Church buffer (7% SDS, 0.25 M Na2HPO4 [pH 7.2], 1 mM EDTA, and 1% BSA) at 65°C for at least 30 min. The cDNA amplicon isolated from the ddRT-PCR gel was used as a template to synthesize 32P-labeled DNA probes for Northern blot hybridization. Twenty-five nanograms of cDNA was labeled using the random-priming technique in the presence of [-32P]dCTP (Amersham) with an oligo-labeling kit (Random Primers Labeling System; Gibco-BRL). Hybridizations were carried out at 60°C for 20 h. The membranes were subsequently washed three times (15 min each) at 65°C in 20 mM Na2HPO4 (pH 7.2) with 4% SDS and subjected to autoradiography at -70°C with a Biomax MS TranScreen-HE intensifying screen (Eastman Kodak) for the appropriate exposure time. Blots were then stripped in 1 mM Tris, 1 mM EDTA, and 0.1-strength Denhardt's reagent (single-strength Denhardt's: 2% BSA, 2% Ficoll, 2% polyvinylpyrrolidone; pH 8.0) for 2 h at 75°C and sequentially reprobed with radiolabeled cDNAs for glucose-regulated protein 94 (GRP94) and 18S rRNA. The GRP94 probe was a hamster cDNA clone generously provided by Dr. Amy S. Lee (USC, Los Angeles, CA). 18S RNA signal was used to determine the relative amounts of RNA loaded into each well and transferred to the membrane [19].
In Situ Hybridization
At the time total RNA was isolated for the ddRT-PCR and Northern blot analyses, some uterine horns were fixed by immersion in 4% paraformaldehyde for 24 h. The horns were then rinsed in two changes of PBS and stored in 70% ethanol until embedded in paraffin and sectioned at 6 µm.
The pGem T-easy vector, containing an amplicon cloned from the differential display experiments, was linearized; and cRNA sense and antisense probes were synthesized in the presence of digoxigenin (DIG)-labeled rUTP (Roche Molecular Biochemicals, Laval, PQ, Canada) according to the manufacturer's instructions. The in situ hybridization technique used has been described in detail previously [20]. Briefly, uterine cross sections were dewaxed in xylene, rehydrated through an alcohol series to double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), digested in proteinase K at 37°C for 5–7.5 min, and acetylated by incubation with freshly prepared 0.1 M triethanolamine and 0.56% (v:v) acetic anhydride at room temperature. After prehybridization at 50°C for 4 h in a 50% formamide, 5-strength SSPE (750 mM NaCl, 50 mM NaH2PO4, 5 mM EDTA, pH 7.4), single-strength Denhardt's solution, the sections were hybridized with 100 ng of cRNA probe and 400 ng of tRNA (in prehybridization solution) under a coverslip for 20 h at 50°C in a sealed humidified chamber. The sections were then washed extensively, treated with RNase, and processed for immunologic detection of the DIG-labeled cRNA probe using anti-DIG antibodies at a dilution of 1:500 (Roche). Color development was allowed to continue until a signal (blue-purple precipitate) was detected with the antisense probe. The color reaction for the sense cRNA-treated slides was terminated at the same time as for the antisense slides. Sections were then mounted using GVA-mount (Zymed Laboratories, San Francisco, CA) and photographed using Northern Eclipse software (Empix Imaging, Mississauga, ON, Canada).
Immunohistochemistry
Uterine cross sections (6 µm thick) were dewaxed in 2 changes of xylene for 10 min and rehydrated through an alcohol series to single-strength PBS. Sections were then incubated in 3% H2O2 (in methanol) for 30 min at room temperature and rinsed in single-strength PBS three times for 5 min each. A blocking step was performed by placing slides in 2% normal rabbit serum (NRS in single-strength PBS) for 1 h and then rinsing in single-strength PBS three times (for 5 min each). Sections were incubated overnight at 4°C with goat anti-GRP78 antibody (antibody N-20; Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:200 in blocking solution (or with goat anti-GRP78 antibody preabsorbed with peptide according to manufacturer's instructions; Santa Cruz Biotechnology). Slides were rinsed again in single-strength PBS and incubated with rabbit anti-goat IgG antibody (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. The slides were again rinsed in single-strength PBS and incubated with ABC reagent (Vecta-stain Kit; Vector Laboratories) for 1 h, rinsed in single-strength PBS, and incubated with diaminobenzidine (Sigma Chemical Company) until a brown color developed. The color reaction for the control (goat anti-GRP78 antibody preabsorbed with control peptide) slides was terminated at the same time as for the slides treated with primary antibody. Slides were counterstained with hematoxylin, dehydrated, cleared with xylene, and mounted under Permount (Fisher Scientific, Pittsburgh, PA). Images were taken using the Northern Eclipse software (Empix Imaging).
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 relative densities of GRP78 and GRP94 signals are presented as the mean relative to a control, as indicated in the figures. The data were analyzed by within-blocks ANOVA, with experiments being considered blocks. Duncan's 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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To identify mRNAs with expression patterns unique to the sensitized endometrium, gene expression during the periimplantation period was compared using the ddRT-PCR technique. RNA from the equivalent of Day 4, 5, or 6 of pseudopregnancy was compared to isolate amplicons unique to the Day 5 or "receptive" endometrium. Two groups of Day 5 animals that received inappropriate doses of E2 on the evening of Day 4 (no E2—low group; and 5.0 µg of E2—high group) were also included in the comparison. This was done in an attempt to identify "receptive-specific" amplicons, as both treatments result in a temporally correct but hormonally nonsensitized endometrium [7]. Amplicons that appeared to be present only in the Day 5 intermediate E2 lanes were isolated and cloned. One such amplicon (Fig. 1) was determined upon sequencing to be glucose-regulated protein 78 (GRP78), which is also called immunoglobulin heavy chain-binding protein (BiP) (previously shown to be the same protein [9]). The isolated cDNA corresponded to bases 347–1214 of rat GRP78 (GenBank accession no. S63521) or bases 1503–2370 of rat BiP (GenBank accession no. M14050).
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Northern blot analysis was used to confirm the expression patterns seen in the ddRT-PCR gel (Fig. 2). The experiment was repeated 3 times, and the up-regulation of GRP78 mRNA in Day 5 intermediate E2 endometrium that was seen in the ddRT-PCR gel was confirmed and found to be statistically significant (P < 0.05).
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Northern Blot Analysis of GRP78 mRNA Expression During Pseudopregnancy and Oil-Induced Decidualization
Further Northern blot analyses allowed the characterization of the temporal expression of GRP78 mRNA over Days 1–7 of pseudopregnancy and during early decidualization (Fig. 3). Again the up-regulation was found on Day 5 of pseudopregnancy, the time of uterine sensitization. Interestingly, GRP78 expression did not appear to increase during the early stages of decidualization, and the up-regulation was restricted to Day 5 (P < 0.01). Days 8–10 stimulated and nonstimulated endometrium were also examined, but a dramatic variation in the signal intensity obtained in the three replicates did not allow characterization of GRP78 mRNA expression during this phase of pseudopregnancy.
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Localization of GRP78 mRNAs in the Pseudopregnant Uterus
In situ hybridization experiments using DIG-labeled cRNA probes for GRP78 verified that an up-regulation of mRNA occurred in Day 5 intermediate E2 endometrium and that it was predominantly within the glandular epithelium (Fig. 4). The up-regulation on Day 5 was consistent with the expression patterns seen within the Northern blots. There was also noticeable localization within the glandular epithelium of Day 5 high E2 endometrium, although to a lesser extent. The absence of staining with the sense probe verified that the signal obtained with the antisense probe was specific for GRP78.
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Localization of GRP78 Protein in the Pseudopregnant Uterus
To determine whether the differential expression of GRP78 mRNA was mirrored by a change in protein expression, immunohistochemistry was done using an antibody specific to the amino terminus of the GRP78 protein (Fig. 5). An increase in GRP78 protein was observed within the endometrial glands of Day 5 intermediate E2 uteri (and Day 5 high E2 uteri, although to a lesser extent), consistent with the observations for the expression of the mRNA. Staining was also evident in the luminal epithelium, although this was not restricted to Day 5 intermediate E2, as staining within this compartment did not change over the periimplantation period. Day 5 low E2 uteri showed a general decrease in staining (in epithelial, stromal, and myometrial compartments) as compared to the other stages. Interestingly, there appeared to be a dramatic up-regulation of GRP78 protein within the myometrium that was specific to the Day 5 intermediate E2 group. In situ hybridizations did not show the concurrent increase in mRNA within the myometrium that was seen within the glandular epithelium. Northern blot analysis did not include myometrial RNA, so quantification of mRNA levels within the myometrium was not performed.
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Northern Blot Analysis of Endometrial GRP94 mRNA Expression During Different States of Uterine Sensitization
GRP94, like GRP78, is a Ca2+-binding ER chaperon found within the ER lumen [11]. GRP94 is almost always observed to be coregulated with GRP78 and is also thought to participate in the protection against various forms of cellular stress (reviewed in [11]). Given that glucose-regulated proteins are generally regulated together, GRP94 mRNA expression was examined during the various states of uterine sensitization used to investigate GRP78 mRNA expression (Fig. 6). The experiment was repeated 3 times, and no statistically significant changes in GRP94 mRNA expression were found (P > 0.6).
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Hormonal Control of GRP78 and GRP94 mRNA Expression in Pseudopregnant Endometrium
Uterine receptivity/sensitization arises as a result of 48 h of P4 priming followed by exposure to a specific concentration of E2 [1]. The induction of GRP78 within the endometrium at the time of uterine sensitization implies that E2 can regulate its expression. To further examine the hormonal regulation of endometrial GRP78 mRNA levels in vivo, animals were ovariectomized, allowed to rest 1 wk, and injected with either vehicle (sesame oil), 1.0 µg of E2, 4 mg of P4, or 1.0 µg of E2 + 4 mg of P4. Uteri were collected 18 h after injection, and total endometrial RNA was isolated and used for Northern blot analysis. The results were unexpected in that either P4 or E2 alone appeared to moderately induce GRP78 mRNA, but given together they did not stimulate expression above that of the control (Fig. 7). By ANOVA, there was a significant interaction (P < 0.05) between E2 and P4 brought about because the effects of E2 and P4 were not additive. It is important to note, however, that the levels of GRP78 mRNA in the endometrium of rats treated with vehicle, P4 alone, E2 alone, or E2 + P4 were low when compared to that seen in Day 5 intermediate E2 endometrial samples (Fig. 7). Hormonal control of GRP94 mRNA expression was also investigated under the same conditions. There were no significant changes in GRP94 mRNA expression as a result of hormone treatments (Fig. 7) or state of uterine sensitization (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The function of GRP78 in the sensitized endometrium is not known. Given GRP78‘s known function as an ER chaperon that assists in the translocation and folding of nascent proteins within the ER, the induction of GRP78 within the glandular epithelium at the time of uterine sensitization may be required to facilitate an increase in protein flux through the ER since there are increased protein synthesis and secretion at this time. A rise in protein synthesis within the endometrium occurs on Day 3 of pregnancy, followed by a decrease on Day 4 and a rise again on Day 5 after which it remains high in implantation sites [21, 22]. It has previously been shown that the normal production of exportable proteins is sufficient to induce the expression of GRP78 and other ER chaperons [12]. In many cell types, the size of the secretory apparatus increases in parallel with an increase in the secretory workload, creating the need for more resident ER proteins [23]. Also, Finn and Martin [24] showed that injection of estrogen to ovariectomized mice following 3 days of progesterone treatment (a hormone regime similar to our protocol for establishing uterine sensitization) leads to maximal protein secretion from the uterine glands within 48 h. However, the idea that GRP78 is induced to allow for an increased secretory load may be an oversimplification. The pattern we have observed for GRP78 during pseudopregnancy does not correspond temporally with the increases in protein synthesis and secretion within the endometrium. Firstly, the ultrastructural changes seen in glandular epithelium, such as increased amounts of rough ER and electron-dense granule accumulation at the apical borders of the cells, do not occur until 48 h after exposure to estrogen, at which time protein secretion is at a maximum [25]. We observed that GRP78 mRNA and protein expression were induced on Day 5 of pseudopregnancy, 18–20 h after E2 administration, and did not remain elevated at 48 h (Day 6 or 7, in the presence or absence of decidualization). Secondly, the pattern of protein synthesis in pseudopregnant rat uteri is somewhat different from that of the pregnant rat in that there is no general increase seen on Day 5 [22]. Thirdly, overall protein content in the rat deciduoma has been shown to rise as much as 70% per day in the second and third days after stimulation [26]. Our experiments did not indicate a sustained increase in GRP78 expression during the early stages of decidualization (see Fig. 3). Together these observations suggest that GRP78 induction on Day 5 of pseudopregnancy is not likely the result of an increased need for ER chaperons due to a general increase in protein synthesis and secretion within the endometrium at this time. They do, however, suggest that an induction of GRP78 within the endometrial glands may have a receptive-specific function.
It has been shown that GRP78 interacts with and is required for the proper folding and secretion of some, but not all, proteins [27]. Since we demonstrated that GRP78 induction was tightly restricted to the Day 5 intermediate E2 endometrium, it is possible that GRP78 is specifically required for the efficient biosynthesis and secretion of proteins involved in the onset of uterine sensitization. The idea that GRP78 is involved in the synthesis and regulation of a group of process-related proteins is not unique, as Beaton et al. [28], in a study to identify lactation-associated and prolactin-responsive proteins in mouse mammary cells, proposed a similar process-specific function for GRP78. They observed an increase in GRP78 expression in primary mouse mammary epithelial cells in response to lactation and in the mouse mammary epithelial cell line COMMA-D when stimulated with prolactin. It was suggested that an induction of GRP78 may function in the processing and secretion of milk proteins. Thus in both cases, lactation and uterine sensitization, GRP78 may play an important role in the regulation and production of a restricted set of proteins required for a secretory event.
Several other functions have been attributed to GRP78 aside from assisting in the folding and processing of proteins in the ER lumen. For example, GRP78 has been implicated in conferring a protective role in cell survival under certain conditions of cellular stress. Glucose starvation, the inhibition of folding, maturation, or export of proteins by agents such as tunicamycin or brefeldin A, perturbation of intracellular Ca2+ levels by Ca2+ ionophores or thapsigargin, and oxygen deprivation have all been shown to induce the expression of GRP78 (reviewed by Little et al. [11]). Also, GRP78 has been shown to play a role in the storage of Ca2+ within the ER lumen and to be involved in intracellular Ca2+ homeostasis [29]. Intracellular Ca2+ signals are important regulators of many cellular events, and their regulation by GRP78 could affect a wide variety of cellular processes. It is uncertain what function, or set of functions, GRP78 may have within the rodent endometrium at the time of uterine receptivity/sensitization.
It is also unclear what is regulating the increase in GRP78 expression at the time of uterine sensitization. The temporal expression pattern we have demonstrated would suggest that estrogen, which is secreted on the evening of Day 4 of pregnancy or pseudopregnancy and is responsible for the onset of uterine receptivity, could be directly responsible for the increase in GRP78 expression. However, when we examined the hormonal regulation of GRP78 mRNA in vivo we found that E2 did not, by itself or together with P4, increase the mRNA expression to the same extent as in the sensitized Day 5 endometrium (Fig. 7). Also, a high dose of E2 given on the evening of Day 4 does not produce a sensitized endometrium [7] and does not induce the expression of GRP78 to the same extent as does the intermediate dose of E2 (Figs. 2–5). These observations indicate that the complex interaction between E2 and P4 required for the onset of a receptive stage is also required for a dramatic increase in GRP78 expression within the rodent endometrium.
The regulation of GRP78 has been widely studied, and an extensive literature has accumulated on the molecules involved in the induction of GRP78 gene expression and on the regulatory elements of the GRP78 promoter. However, most of the studies have focused on the induction of GRP78 under conditions of "stress," such as Ca2+ depletion and inhibition of protein folding and maturation within the ER [30–39]. There are few examples of induction of the glucose-regulated proteins in response to stimuli that do not induce cellular stress. Some evidence, however, has accumulated demonstrating the regulation of GRPs by some growth factors through a pathway distinct from that of stress induction [40].
Oxygen deprivation, a form of cellular stress, has been shown to induce GRP78 expression [41]. Intrauterine oxygen tension in the hamster decreases under estrogenic stimulation, during proestrus [42], and at the time of blastocyst formation and implantation [43], perhaps due to luminal closure. This represents a possible mechanism whereby GRP78 could be induced within the rodent endometrium through the stress response pathway.
However, on the basis of the results we have observed on the expression of GRP94 mRNA, it is likely that the induction of GRP78 at the time of uterine sensitization is the result of regulation through a pathway distinct from the cellular stress response. GRP94 is usually coregulated with GRP78 in a general response to cellular stresses (reviewed in [11]). Our data demonstrate that GRP94 is not induced concurrently with GRP78 during uterine sensitization (Fig. 6). This suggests that the attainment of a receptive state does not likely activate a cellular stress pathway and elevate GRPs but that it induces GRP78 mRNA expression via a novel, uncharacterized pathway. GRP expression via a pathway distinct from the unfolded protein response (or stress response) has been previously demonstrated [40]. However, this signaling pathway induced the expression of both GRP78 and GRP94 mRNAs. Within the pseudopregnant rat endometrium, GRP78 mRNA is induced at the time of maximal sensitivity for the decidual cell reaction independently of GRP94 mRNA. Studies are currently under way to identify the mechanisms involved in the induction of GRP78 within the rodent endometrium at the time of uterine sensitization.
In summary, we have demonstrated the induction of GRP78 within the uterine glandular epithelium at the optimal time of sensitization for blastocyst implantation. Its expression is subsequently down-regulated once the endometrium has become refractory or begins the process of decidualization. Also, GRP78 appears to be regulated independently of another glucose-regulated protein, GRP94, suggesting that it may have a specific and an as yet undefined function during uterine sensitization. Also, this study demonstrates a novel system for the induction of GRP78, although this remains to be fully characterized.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Medical Research Council of Canada, Grant MT-10414. 
2 Correspondence: T.G. Kennedy, Department of Physiology, The University of Western Ontario, London, ON, Canada N6A 5C1. FAX: 519 661 3827; tom.kennedy{at}physiology.uwo.ca
Accepted: December 6, 1999.
Received: August 23, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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