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BOR - Papers in Press, published online ahead of print October 20, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033837
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BIOLOGY OF REPRODUCTION 72, 700–706 (2005)
DOI: 10.1095/biolreprod.104.033837
© 2005 by the Society for the Study of Reproduction, Inc.

Inhibition of the Beta-Catenin Signaling Pathway in Blastocyst and Uterus During the Window of Implantation in Mice1

Jing Li3,4, Jian V. Zhang5, Yu-Jing Cao3, Jia-Xi Zhou3, Wei-Min Liu3, Xiu-Jun Fan3, and En-Kui Duan2,3

State Key Laboratory of Reproductive Biology,3 Institute of Zoology, Chinese Academy of Sciences, Beijing, 100080, China Graduate School of the Chinese Academy of Sciences,4 Beijing 100039, China Division of Reproductive Biology,5 Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Beta-catenin, the mammalian homolog of Drosophila armadillo protein, was first identified as a cadherin-associated protein at cell-cell junctions. Another function of beta-catenin is the transduction of cytosolic signals to the nucleus in a variety of cellular contexts, which usually are elicited by the active form of beta-catenin. The aim of the present study was to examine the potential role of active beta-catenin in the mouse embryo and uterus during embryo implantation. Active beta-catenin was detected differentially in mouse embryos and uteri during the peri-implantation period. Aberrant activation of beta-catenin by LiCl, a well-known glycogen synthase kinase-3 inhibitor, significantly inhibited blastocyst hatching and subsequent adhesion and outgrowth on fibronectin. Results obtained from pseudopregnant and implantation-delayed mice imply an important role for implanting blastocysts in the temporal and spatial changes of active beta-catenin in the uterus during the window of implantation. Collectively, these results suggest that the beta-catenin signaling pathway is inhibited in both blastocyst and uterus during the window of implantation, which may represent a new mechanism to synchronize the development of preimplantation embryos and differentiation of the uterus during this process.

embryo, implantation, pregnancy, signal transduction, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Beta-catenin, the mammalian homolog of Drosophila armadillo protein, was initially described as a protein that interacts with the cytoplasmic tail of E-cadherin [1]. In addition to its role in cadherin-mediated cell-cell adhesion, beta-catenin also serves a noncadherin-dependent signal transduction function in a variety of cellular contexts. In the canonical Wnt pathway, binding of some Wnt proteins to their partner Frizzled receptors inhibits the activity of glycogen synthase kinase-3ß (GSK3ß), a negative regulator of Wnt/beta-catenin signaling. When activated, GSK3ß phosphorylates APC (adenomatous polyposis coli) and Axin, increasing their binding affinities toward beta-catenin, which was then marked for proteasome-mediated degradation by N-terminal phosphorylation. The GSK3ß inactivation in response to Wnt negates formation of this complex, subsequently leading to accumulation of a cytoplasmic pool of beta-catenin, which usually is considered to be the active form of beta-catenin. Active beta-catenin then translocates into the nucleus, where it binds to transcription factors of the T-cell factor/lymphoid-enhancing factor (Tcf/Lef) family to activate the transcription of a specific subset of genes [2, 3]. Recently, a new monoclonal antibody, {alpha}-active beta-catenin ({alpha}ABC), has been shown to permit visualization of active beta-catenin generation by the canonical Wnt pathway during murine embryogenesis [4]. Thus, {alpha}ABC may be instrumental as a tool for outlining those signaling events that activate beta-catenin-mediated signal transduction.

The process of implantation involves complex interactions between the blastocyst and the uterus. Synchronized development of the embryo to the blastocyst stage, differentiation of the uterus to a receptive state, and cross-talk between the blastocyst and uterine luminal epithelium are essential to the implantation process [5]. The canonical Wnt/beta-catenin signaling pathway exhibits pivotal roles in cell proliferation, differentiation, and epithelial-mesenchymal communication [6]. The important roles of the Wnt/ beta-catenin signaling pathway in postimplantation embryo development, including the specification of cell fate, induction of the body axis, and determination of embryonic patterning, have been investigated extensively in both invertebrates and vertebrates [6], but recently, expression of Wnt-3a and Wnt-4 mRNA in mouse embryos from the 4- to 8-cell stages to the blastocyst stage indicates a potential role for the Wnt/beta-catenin signaling pathway in preimplantation embryo development and during the window of implantation [7].

In the adult mouse uterus, several Wnt genes have been identified, and their expression seemed to fluctuate during the estrous cycle [8]. Although no significant menstrual cycle dependence of the Wnt ligands (except Wnt-3), receptors, or downstream effectors was observed in human endometrium, regulators of the canonical Wnt/beta-catenin signaling pathway, Dkk-1 and FrpHE, showed dramatic changes during the window of implantation [9, 10]. Furthermore, some of the target genes for Wnt/beta-catenin signaling, such as Hoxa-10 [11], Hoxa-11 [11], PPAR-{delta} (peroxisome proliferator-activated receptor-{delta}) [12], and COX-2 (cyclooxygenase-2) [12], are important in decidualization of the endometrium and in implantation in both mice and humans. It is possible that a Wnt-signaling dialog between the epithelial and stromal components exists during the window of implantation. However, growth factors, such as epidermal growth factor [13, 14] and hepatocyte growth factor [13, 15, 16], which appear to have an important functional role in mammalian implantation, also showed elevated cytoplasmic beta-catenin- and enhanced beta-catenin/Lef-dependent transcription in other tissues. Implantation of the embryo is dependent on multiple signal pathways via a complex network to prompt blastocyst implantation into the maternal endometrium [17], and the fact that active beta-catenin resides at the hub of several major signaling pathways suggests that a very complicated mechanism exists that regulates the activity of active beta-catenin during the window of implantation.

In the present study, to investigate the potential role of beta-catenin signaling pathway in embryo implantation, we examined the spatiotemporal distribution of active beta-catenin in the mouse embryo and uterus during the peri-implantation period using {alpha}ABC, a novel antibody specific for active beta-catenin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Adult (virginal) mice (weight, 22–25 g) of the outbred Kunming white strain were purchased from the Experimental Animal Center, Institute of Zoology, Chinese Academy of Sciences, and raised at 25°C in a constant photoperiod (14L:10D). The Guidelines for the Care and Use of Animals in Research were followed. Mice were allowed free access to water and food. Virgin female mice were mated with fertile or vasectomized males of the same strain. The morning of finding a vaginal plug was designated as Day 1 of pregnancy or pseudopregnancy. Pregnancy on Days 1–4 was confirmed by recovering embryos from the reproductive tracts. Implantation sites ensured pregnancy on Days 5–8. Uteri from Days 1–8 of pseudopregnancy were collected for further analysis.

To induce delayed implantation, pregnant mice were ovariectomized at 0830–0900 h on Day 4 of pregnancy. They were assigned randomly into five groups and treated with different combinations of steroid hormones from Days 5–7. All steroids were dissolved in sesame oil and injected s.c. Mice in the control group were injected with sesame oil (0.1 ml/mouse; Sigma) as a control. Mice in the estradiol-17ß (E2) group, progesterone (P4) group, and E2+P4 group were injected with E2 (25 ng/mouse; Sigma), P4 (1 mg/mouse; Sigma), and a combination of E2 and P4, respectively. In the (P4)2+E2+P4 group, mice were injected with P4 (1 mg/mouse) to maintain delayed implantation from Days 5 to 7, and on Day 7, E2 (25 ng/mouse) was given to P4-primed. delayed-implantation mice to terminate delayed implantation. All mice were killed for uteri collection 12 h after steroid hormone treatments. In the (P4)2+E2+P4 group, only uteri from which embryos had been recovered were collected, and uteri without embryos were used as controls.

Embryos: Collection, Culture, and Treatment

Preimplantation embryos at different developmental stages were flushed from the oviduct or uterus at 0900 h on Days 2–4 of pregnancy. Some blastocysts on Day 4 were further cultured in Ham F-12 (Gibco, Rockville, MD) with 0.4% BSA (Sigma) until they hatched through the zona pellucida. Blastocysts that were not hatched in this manner were freed of the zona pellucida using 0.4% pronase (Sigma) in Ham F-12. All embryos were then fixed for 30 min in freshly prepared 4% paraformaldehyde (Sigma) for indirect immunofluorescence.

Some blastocysts flushed from the uterus at 0900 h on Day 4 were cultured in 0.4% BSA in Ham F-12 with or without LiCl and/or 5 mM antisense or sense oligonucleotides (ODN) against beta-catenin (antisense beta-catenin, 5'-GGAGTTTAACCACAACAGGCAGTCC-3' [18]; sense beta-catenin, 5'-GGACTGCCTGTTGTGGTTAAACTCC-3'). At the termination of culture after 24 h, the percentage of blastocysts hatching from the zona pellucida was recorded.

In vitro attachment and outgrowth assays for assessment of implantation were performed according to the methods established by our group [19]. In brief, after 24 h of culture, hatched blastocysts in the control group and the 25 mM LiCl group were transferred to 0.4% BSA in Ham F-12 with or without antisense or sense beta-catenin ODN. Culture dishes were precoated with 10 µl of fibronectin (FN; 1 µg/µl; Sigma). The attachment or outgrowth was observed at 24 or 48 h using phase-contrast microscopy.

Immunohistochemistry

Mouse uteri were immediately cut into small pieces, fixed in Bouin solution, dehydrated, and embedded in paraffin. After sections (thickness, 5 µm) were cut, deparaffinized, and rehydrated, endogenous peroxidase activity was blocked by incubating the sections in 1.5% peroxide in methanol for 20 min. The sections were then boiled in citrate buffer (0.01 mol/ L, pH 6.0) for 15 min and cooled slowly. Nonspecific binding was blocked in 10% normal horse serum in PBS for 40 min. Then, the sections were incubated with {alpha}ABC (1:300; catalog no. 05-601; Upstate Biotechnology, Waltham, MA) overnight at 4°C. After washing in PBS, the sections were incubated with biotin-conjugated secondary horse anti-mouse antibodies (Zhongshan Biotechnology, Beijing, China) for 45 min at 37°C. The sections were then washed in PBS and incubated with horseradish peroxidase streptavidin (Zhongshan Biotechnology) for another 45 min at 37°C. After rinsing in PBS, the primary antibody was detected with 3,3'-diaminobenzidine solution (Zhongshan Biotechnology). For some sections, primary antibodies were replaced with mouse preimmune immunoglobulin (Ig) G as a negative control.

Western Blot Analysis

Proteins obtained from uterine lysates were boiled in SDS/ß-mercaptoethanol sample buffer, and 50-µg samples were loaded onto each lane of 10% polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were blotted onto nylon membranes (Amersham Biosciences) by electrophoretic transfer in 25 mmol/L of Tris and 192 mmol/L of glycine buffer at pH 8.3. Beta-catenin proteins were detected by probing the blot first with {alpha}ABC and pan-beta-catenin (catalog no. RB-1491-P0; NeoMarkers Biotechnology, Fremont, CA) antibodies, followed by alkaline phosphatase-conjugated horse anti-mouse IgG ({alpha}ABC) and goat anti-rabbit IgG (pan-beta-catenin), respectively, with final visualization of positive signals by BCIP/NBT methods (Zhongshan Biotechnology).

Indirect Immunofluorescence

After fixation in 4% paraformaldehyde, embryos were washed three times in PBS and permeabilized for 20 min in PBS containing 0.2% Triton X-100 (Sigma) at room temperature. After rinsing several times in PBS, embryos were incubated in 5% BSA for 45 min at room temperature to block nonspecific binding of the antibodies. The BSA solution was then aspirated with filter paper, and embryos were incubated with {alpha}ABC diluted 1:100 in PBS at 4°C overnight. After rinsing in PBS, embryos were incubated in fluorescein isothiocyanate-conjugated secondary antibody (Zhongshan Biotechnology) at 37°C for 1 h, then rinsed in PBS. Nuclei were stained with 0.01 mg/ml of propidium iodide (Sigma) for 10 min. Embryos were viewed under a laser-scanning confocal microscope (Leica, Heidelberg, Germany). For some embryos, pan-beta-catenin antibody (also diluted 1:100 in PBS) was used as a positive control and mouse preimmune IgG as a negative control. As a control for nonspecific staining by the second antibody, staining omitting the primary antibody also was performed.

Statistics

One-way ANOVA was used for statistical evaluation of the data, and significance of the differences between groups was determined by the Scheffé test as appropriate, with P < 0.05 considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dynamics of Active Beta-Catenin in Mouse Embryo and Uterus During the Peri-Implantation Period

To study the role of active beta-catenin in embryo implantation, we first examined its temporal and spatial distribution in both the embryo and the uterus during the peri-implantation period.

It has been shown previously that the bulk of beta-catenin accumulation occurs in the cytoplasm from 2-cell embryos to the morula stage, but no evidence of nuclear beta-catenin staining has been observed [20], indicating that the beta-catenin signaling pathway may not be activated during this period. Consistent with these earlier observations, in the present study active beta-catenin was not detected in either the cytoplasm or the nucleus from 2- to 16-cell embryos (Fig. 1A), but at the late morula and early blastocyst stages, we detected a granular distribution of active beta-catenin in the cytoplasm and the nucleus (Fig. 1, B and C). When fully expanded blastocysts hatched from the zona pellucida for implantation, the signal for active beta-catenin disappeared (Fig. 1D). Following successful embryo implantation into the endometrium, active beta-catenin was still inhibited until Day 7 of pregnancy, when strong signals of active beta-catenin were detected in embryos (Fig. 2, E, G, and I). However, before the activation of beta-catenin in embryos, active beta-catenin was first detected in invasive trophoblast on Day 6 of pregnancy, and its expression became stronger on Day 7 of pregnancy (Fig. 2, G and I).



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  		FIG. 1. Immunofluorescence of mouse cleavage-stage embryos with {alpha}ABC. Green represents active beta-catenin staining, and red indicates nuclear staining. Yellow coloration represents an overlap of green and red. A) 16-Cell mouse embryo. B) Late morula stage. C) Early blastocyst stage. D) Hatched blastocyst from the zona pellucida. E) Hatched blastocyst following treatment with 25 mM LiCl for 24 h. F) Positive control: distribution of total beta-catenin in morula as detected by pan-beta-catenin antibody. G) Positive control: expression of total beta-catenin in hatching blastocyst. H) Negative control: primary antibodies replaced by mouse IgG. I) Negative control: primary antibodies omitted. Bar = 20 µm



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  		FIG. 2. Immunostaining of active beta-catenin protein in mouse uterus on Days 3 (A), 4 (B), 5 (D and E), 6 (G and H), 7 (I and J) of pregnancy, respectively. We detected no difference of active beta-catenin in stroma on Days 4 (C) and 5 (F) of pseudopregnancy. Staining of active beta-catenin after terminating delayed implantation following (P4)2+E2+P4 treatment for 12 h (K) is also shown, as is the negative control (L). Cross-sections were used for all samples except Day 5, when longitudinal sections were used. de, Decidua; em, embryo; ge, glandular epithelium; le, Luminal epithelium; s, stroma; tr, trophoblast. Bar = 100 µm

Concurrent with these findings, active beta-catenin also showed unique expression patterns in the uterus during the peri-implantation period. Although no difference was detectable in the levels of total beta-catenin from Day 3 to Day 7 of pregnancy, active beta-catenin in the uterus showed dramatic changes during this period. Following a gradual decrease during the first 4 days, with the lowest level occurring at Day 4, dramatic increases of active beta-catenin protein were observed on Day 5, and this high level was maintained until Day 7 (Fig. 3, A and C). The immunostaining results are consistent with what was observed by Western blot analysis, in that active beta-catenin in stromal cells reached the lowest levels on Day 4 (Fig. 2B), but following implantation on Day 5 and thereafter, the staining of active beta-catenin became more intense and was restricted primarily to undifferentiated stromal cells (Fig. 2, D, H, and J).



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  		FIG. 3. Different expression patterns of active beta-catenin in uterus samples between Day 3 and Day 7 of pregnancy (A) and pseudopregnancy (B). Total beta-catenin was used as a positive control to determine total levels of membrane-associate beta-catenin and active beta-catenin. An antibody against ß-actin was used in the same Western blot as a loading control. Densitometric analysis of the Western blots regarding active beta-catenin (C) is also shown. Results are presented as the mean ± SEM of three replicates. ***Significant difference on Day 5 of pregnancy compared to Day 4 (P < 0.001)

Effects of Active Beta-Catenin on Blastocyst Hatching and Adhesion and Outgrowth on FN

Given the disappearance of active beta-catenin in hatched blastocysts and in embryos on Days 5 and 6 of pregnancy, we speculated that abnormal activation of beta-catenin signaling may disturb the process of blastocyst hatching and subsequent embryo implantation. The Wnt-induced stabilization of cytoplasmic beta-catenin can be simulated by LiCl, which mimics Wnt by inhibiting the serine kinase activity of GSK3 [4]. As shown in Figure 4A, LiCl incubation for 24 h was able to significantly decrease the percentage of blastocysts hatching in a dose-dependent manner. Indeed, compared with the normal hatched blastocysts (Fig. 1D), LiCl had the capacity to restore the accumulation of beta-catenin in the cytoplasm and nuclei of hatched blastocysts in the 25 mM group (Fig. 1E). Given that the percentage of blastocysts hatching was affected only minimally following treatment with 25 mM LiCl for 24 h (Fig. 4A) and that active beta-catenin clearly was detected in the nuclei of those hatched blastocysts, these blastocysts were transferred to normal cultured media in wells coated with 10 µl of FN to determine the effects of active beta-catenin on embryo implantation in vitro. As shown in Figure 4B, after 24 and 48 h of coculture, the percentage of embryos attaching and their outgrowth on FN decreased dramatically.



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  		FIG. 4. LiCl inhibits blastocyst hatching (A) and attachment and outgrowth on FN (B). A) Percentage of blastocysts hatching after 24 h of culture, with indicated concentrations of LiCl in Ham F-12 containing 0.4% BSA. The results are shown as the mean ± SEM for three independent experiments, and each group contained at least 50 blastocysts. **P < 0.01, ***P < 0.001. B) Percentage of attachment and outgrowth of blastocysts on FN. Blastocysts were treated for 24 h with the indicated concentrations of LiCl, and then hatched blastocysts were transferred into Ham F-12 containing 0.4%BSA. The culture dish was precoated with FN. After 24 and 48 h of culture, the percentage of attachment and outgrowth was determined as discussed in Materials and Methods. At least 30 blastocysts were used, and results are shown as the mean ± SEM of three replicates. **P < 0.01

Antisense ODN transfection of beta-catenin reduces beta-catenin expression levels and, specifically, its transcriptional activity [18]. To verify that the impairment of LiCl on blastocyst hatching and attachment and outgrowth on FN results from the activation of beta-catenin in the nucleus, 5 mM antisense beta-catenin ODN was added directly in 0.4% BSA in Ham F-12 to reduce beta-catenin expression levels. Surprisingly, it did not reverse the inhibition of LiCl on blastocyst hatching. Therefore, we speculate that the zona pellucida provides a barrier that prevents the ODN from entering into the blastocysts (data not shown). However, when those hatched blastocysts in the 25 mM group were transferred into 0.4% BSA in Ham F-12 supplemented with 5 mM antisense ODN, percentages of adherence and outgrowth of blastocysts on FN after 24 and 48 h of culture were rescued to an extent similar to that exhibited by the control group and differed significantly from blastocysts treated with the same dose of sense beta-catenin ODN (P < 0.05) (Fig. 5). Furthermore, in those blastocysts treated with antisense beta-catenin ODN, we actually detected a significant decrease of beta-catenin mRNA and total protein compared with those blastocysts in the control group and those treated with sense ODN (data not shown).



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  		FIG. 5. Antisense ODN of beta-catenin partly rescues the inhibitory effects of LiCl on attachment and outgrowth of blastocysts on FN. After pretreatment with 25 mM LiCl for 24 h, hatched blastocysts were transferred into 0.4% BSA in Ham F-12 into dishes precoated with FN together with the indicated concentrations of antisense or sense ODN. At least 30 blastocysts were used, and results are shown as the mean ± SEM of three replicates. *P < 0.05, ***P < 0.001

Dynamics of Active Beta-Catenin in the Uterus of Pseudopregnant and Implantation-Delayed Mice

Our results in mouse uteri during the peri-implantation period suggest that active beta-catenin in the uterus is influenced by implanting blastocysts. Uterine events that occur during the peri-implantation period also can be induced in implantation-delayed or pseudopregnant mice [21], suggesting that these mice may be useful for studying the effects of activated blastocysts on temporal and spatial changes of active beta-catenin in the uterus during the peri-implantation period. As shown in Figure 3, B and C, neither total beta-catenin nor active beta-catenin showed differences among uterus samples from Day 3 to Day 7 of pseudopregnancy. Consistent with what was observed by Western blot analysis, active beta-catenin localized in stromal cells showed no differences during this same period (Fig. 2, C and F). However, as shown in Figure 6, although the protein level of active beta-catenin was slightly increased in the implantation-delayed mice under the E2+P4, E2, and P4 treatments, it was dramatically decreased following the treatment of (P4)2+E2+P4 (P < 0.05). These results are further supported by the lower staining of active beta-catenin in the uterus after terminating the delayed implantation following (P4)2+E2+P4 treatment for 12 h, as shown in Figure 2K.



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  		FIG. 6. Western blots of active beta-catenin proteins in uteri of implantation-delayed mice. Total beta-catenin was used as a positive control to determine total levels of membrane-associate beta-catenin and active beta-catenin. An antibody against ß-actin was used as a loading control. The different hormone treatments were as discussed in Materials and Methods. A densitometric analysis of the Western blot also is shown. Results are presented as the mean ± SEM of three replicates. Lane 1: control group; lane 2: E2+P4 group; lane 3: E2 group; lane 4: P4 group; lane 5: (P4)2+E2+P4 group. *Significant decrease of active beta-catenin following (P4)2+E2+P4 treatment for 12 h compared with the control group (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study showed that active beta-catenin was regulated differentially in both the embryo and the uterus during the window of implantation. At the late morula and early blastocyst stages, the accumulation of active beta-catenin in the cytoplasm and nucleus indicates that beta-catenin signaling may be involved in blastocyst formation. The importance of E-cadherin/catenin cell-adhesion complexes for compaction and blastocyst formation during early mouse embryonic development has been well investigated in previous studies [22]. Although Lef-1 has shown no expression [20] in embryos until Day 7.5 of pregnancy, the beta-catenin signaling pathway may still function in this process, because beta-catenin can bind other transcription factors separate from those of the Tcf/Lef family to induce expression of some of the target genes [23].

The process of implantation involves preimplantation embryos developing to the blastocyst stage and blastocyst hatching from the zona pellucida to establish reciprocal interactions between the trophectoderm and uterine luminal epithelium. In the present study, we found, surprisingly, that active beta-catenin disappeared as soon as blastocysts escaped from the zona pellucida and that this inhibition was maintained until Day 7 of pregnancy, when high levels of active beta-catenin were observed in embryos and the trophoblast. These results suggest that beta-catenin signaling in blastocysts may be inhibited during embryo implantation. Beta-catenin knock-out mice have severe gastrulation defects and die at 7 days postcoitus [24], and the intense staining of active beta-catenin in embryos and trophoblasts from Day 7 onward verifies the pivotal role of activated beta-catenin signaling pathway in gastrulation, embryo development, and trophoblast invasion.

To further support this assessment, LiCl, an agent that promotes the accumulation of beta-catenin in the cytoplasm and nucleus, was used to study the effect of activated beta-catenin signaling pathway on blastocysts during implantation. In the present study, LiCl inhibited blastocyst hatching in a dose-dependent manner. Although the percentage of blastocysts hatching in the 25 mM group showed no statistical difference from that in the control group, accumulation of active beta-catenin in the cytoplasm and nucleus of those hatched blastocysts demonstrates that the beta-catenin signaling pathway may be activated. Then, when these normal blastocysts were used for in vitro attachment and outgrowth on FN, their attachment and outgrowth decreased significantly even in normal cultural medium without any use of LiCl. In the experiment, active beta-catenin also was detected localizing at cell-cell borders of blastocysts in the 25 mM group (Fig. 1E). In tissue culture cells, cadherin had a potential role in sequestering signaling beta-catenin at the plasma membrane [25]. Thus, after dealing with LiCl for 24 h, the localization of active beta-catenin at the plasma membrane suggests that the same regulation mechanism of E-cadherin in beta-catenin signaling pathway also existed in blastocysts.

Treating 2- or 8-cell mouse embryos with LiCl induces delayed defects beginning at gastrulation, but these do not arise through the beta-catenin/Lef-1 pathway and have no apparent effects on the preimplantation stage [26, 27]. This finding is not in agreement with our observations in the present experiment, but discrepancies may arise from methodological differences concerning either embryonic development stages (blastocysts vs. 2- or 8-cell embryos) or concentrations and durations of treatment with LiCl (25, 35, 45, and 55 mM LiCl for 24 h vs. 300 mM LiCl for 5 min). Whether blastocysts treated with LiCl can be induced to generate axial defects at gastrulation needs to be studied further, but our results suggest that after treatment with 25 mM LiCl, something must have been changed by the activation of the beta-catenin signaling pathway, which resulted in the disturbance of implantation. This could be regarded as a natural selection mechanism to avoid abnormal development and inferior pregnancy outcomes [28].

Aside from GSK3, other well-documented pharmacological targets of LiCl include the phosphoinositol phosphatases, exerting effects that can be rescued indirectly by myo-inositol supplementation [29]. In this regard, we have observed that the addition of myo-inositol to the culture medium does not reverse the inhibitory effects of LiCl on blastocyst hatching or the subsequent defects in adherence and outgrowth on FN (data not shown). However, the inhibitory effects of LiCl on blastocyst attachment and outgrowth on FN could be reversed, in part, by the addition of antisense beta-catenin ODN, a specific agent to inhibit beta-catenin-dependent transcription by reducing beta-catenin expression levels. These results further verify that the inhibitory effect of LiCl on blastocysts during implantation is exerted, at least in part, through deactivation of the GSK3/beta-catenin degradation pathway as opposed to the interference of inositol metabolism.

In the uterus, beta-catenin also showed unique changes during the peri-implantation period. Consistent with what was observed by Western blot analysis, active beta-catenin detected in stromal cells reached a minimum on Day 4, but following implantation on Day 5 and thereafter, the staining of active beta-catenin became more intense and was restricted primarily to undifferentiated stromal cells. Recently, another study has shown the same expression pattern for sFRP4, a regulator of the Wnt/beta-catenin signaling pathway, suggesting that the temporal and spatial distribution of active beta-catenin in mouse uteri may be regulated by sFRP4 during the peri-implantation period [30]. However, implanting blastocysts seemed to be major contributors to the regulation of active beta-catenin, because its expression showed no apparent differences between Day 3 to Day 7 of pseudopregnancy. Furthermore, in implantation-delayed mice, active beta-catenin protein levels decreased dramatically following termination of the delayed implantation after 12 h, whereas the same change could not be detected in uteri without blastocysts (data not shown). These results further underscore the importance of activated blastocysts in regulating the dynamics of active beta-catenin in the uterus during the window of implantation. In addition, although total beta-catenin protein levels showed no statistical difference in pregnant and implantation-delayed mouse uteri, small variations could still be detected among samples. Overexpression of beta-catenin can activate the beta-catenin signaling pathway, which then induces duplication of a complete secondary axis in early Xenopus embryos [31] and early zebrafish embryos [32]. In mouse embryo stem cells, increased beta-catenin protein levels induced Lef-1 protein and a beta-catenin/Lef-1 complex to be translocated to the nucleus [26]. The low expression of beta-catenin on Day 4 of pregnancy and in the (P4)2+E2+P4 group suggests that some molecules reproduced by activated blastocysts could regulate, at least somewhat, the expression of total beta-catenin proteins. However, after being analyzed statistically, our results showed that the beta-catenin signaling pathway more likely is regulated by a posttranscriptional mechanism. How activated embryos regulate dynamic changes of active beta-catenin in the mouse uterus during the window of implantation needs to be studied further.

In conclusion, the present study revealed the unique role of active beta-catenin in both mouse embryos and uteri during the peri-implantation period. When blastocysts hatch through the zona pellucida, obtaining the ability for endometrium attachment during embryo implantation, inhibition of the beta-catenin signaling pathway in both the embryo and the uterus may represent a new mechanism to synchronize development of the preimplantation embryo and differentiation of the uterus.


   FOOTNOTES
 
1 Supported by the Special Funds for Major State Basic Research Project (G1999055903) and Funds from NSFC (30170112). Back

2 Correspondence: En-Kui Duan, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. FAX: 86 10 62631831; duane{at}ioz.ac.cn Back

Received: 29 June 2004.

First decision: 15 July 2004.

Accepted: 5 October 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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