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Wnt genes are involved in critical developmental and growth processes. The present study comprehensively analyzed temporal and spatial alterations in Wnt and Fzd gene expression in the mouse uterus during peri-implantation of pregnancy. Expression of Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd2, Fzd4, and Fzd6 was detected in the uterus during implantation. Wnt4 mRNA was most abundant in the decidua, whereas Wnt5a mRNA was restricted to the mesometrial decidua during decidualization. Wnt7a, Wnt7b, and Wnt11 mRNAs were abundantly detected in the endometrial epithelia. The expression of Wnt7b was robust in the luminal epithelium (LE) at the implantation site on Gestational Day 5, whereas Wnt11 mRNA disappeared in the LE adjacent to the embryo in the antimesometrial implantation chamber but remained abundant in the LE. Wnt16 mRNA was localized to the stroma surrounding the LE on Day 4 and remained in the stroma adjacent to the LE but not in areas undergoing the decidual reaction. Fzd2 mRNA was detected in the decidua, Fzd4 mRNA was in the vessels and stroma surrounding the embryo, and Fzd6 mRNA was observed in the endometrial epithelia, stroma, and some blood vessels during implantation. Ovarian steroid hormone treatment was found to regulate Wnt genes and Fzd receptors in ovariectomized mice. Especially, single injections of progesterone stimulated Wnt11 mRNA, and estrogen stimulated Wnt4 and Wnt7b. The temporal and spatial alterations in Wnt genes likely play a critical role during implantation and decidualization in mice.
The adult uterus undergoes dynamic cellular and molecular changes during pregnancy. Successful establishment of pregnancy in rodents is the result of reciprocal interactions between the implantation-competent blastocyst and receptive uterus [1, 2]. Implantation requires the coordinated effects of estrogen and progesterone (P4). In rodents, estrogen stimulates proliferation and differentiation of luminal epithelium (LE) and glandular epithelium (GE), whereas both estrogen and P4 are required in the process of stromal differentiation [3, 4]. After mating, the uterus is under the influence of preovulatory estrogen on Day 1 (D1; vaginal plug), with heightened LE proliferation. Rising P4 levels secreted from the newly formed corpora lutea initiate stromal cell proliferation from D3. Stromal cell proliferation is further stimulated by ovarian estrogen on the morning of D4 [1, 2]. On D5, attachment between LE and blastocyst trophectoderm is initiated, and endometrial vascular permeability develops solely at the site of the blastocyst while stromal cells continue to proliferate. The coordinated effects of estrogen and P4 during the window of uterine receptivity result in the cessation of uterine cell proliferation and differentiation [2, 3, 5]. On D6 and D7, the implantation process is advanced, with extensive stromal cell decidualization . The uterus must be in a receptive state when blastocysts are ready to initiate implantation. This requires coordinated integration of various signaling pathways between the blastocyst and the uterus. Molecular and genetic evidence indicates that locally produced signaling molecules, including cytokines, growth factors, homeobox transcription factors, lipid mediators, and morphogens, together with ovarian hormones, serve as autocrine, paracrine, and juxtacrine factors that mediate uterine receptivity to blastocyst implantation [3, 5, 7, 8].
Wnt (wingless-type MMTV integration site family member) genes are related to the Drosophila segment polarity gene wingless (wg) and encode a large group of highly conserved secreted glycoproteins. In humans and mice, the Wnt family encodes a group of 19 highly conserved secreted signaling molecules that are critical regulators of cell fate, growth, and differentiation, as well as cell-cell interactions . Autocrine or paracrine signaling by WNT involves a family of 10 frizzled receptors (FZDs) , which are seven-transmembrane G protein-coupled receptors that possess an extracellular cysteine-rich domain for WNT binding . Although it is established that WNT signaling is crucial for early developmental processes  that include development of the female reproductive tract, the importance of WNT signaling in uterine receptivity during pregnancy has only recently been examined.
Many WNT genes are expressed in the human endometrium during the proliferative and secretory phases of the menstrual cycle , and the canonical WNT signaling pathway is implicated in trophoblast differentiation in humans , sheep , and cattle . Wnt4 has been reported to be an important regulator of blastocyst implantation in the mouse uterus , and Wnt4 expression increases with decidualization in both mice and humans [18, 19]. The key mediator of canonical WNT signaling, CTNNB1, is an important regulator of blastocyst implantation  and decidualization . Of particular note, we found that WNT7A, which is expressed only in the endometrial LE and superficial ductal glands of the uterus, stimulated trophectoderm cell proliferation, and WNT5A, abundantly expressed in the stroma, accelerated trophectoderm cell migration in sheep . Thus, WNT signaling is likely a conserved regulator of blastocyst-endometrial interaction in mammals. However, the majority of WNT family genes have not been investigated during the peri-implantation period of pregnancy.
Our working hypothesis is that Wnt genes expressed in the receptive uterus during implantation regulate establishment and maintenance of pregnancy via autocrine and/or paracrine actions through FZD receptors. As a first step in testing this hypothesis, we determined temporal and spatial alterations in the localization of Wnt and Fzd mRNA in the mouse uterus during the implantation. Effects of ovarian steroid hormones on the Wnt and Fzd mRNA levels and localization were also examined.
All procedures were performed in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and were approved by the Institutional Animal Care and Use committees of Texas A&M University and Southern Illinois University. Adult virgin CD1 female mice (6–8 wk old) were obtained from Charles River Laboratories (Wilmington, MA) and mated with fertile males of the same strain to establish pregnancy. All mice were housed in a temperature-controlled room (21°C to 22°C) in a 12L:12D cycle and allowed free access to food and water. In study 1, female mice (8–10 wk old) were mated with fertile males, and mating was confirmed by the presence of a vaginal plug (D1 = vaginal plug). Uteri were collected at 0900 h on Days 1, 2, 3, 4, 5, 6, or 7 after mating. Segments of the uterus containing implanting embryos (implantation site) or not containing implanting embryos (nonimplantation site) were dissected. Implantation sites on D5 and D6 were visualized following intravenous injection (0.1 ml/mouse) of 1.0% Chicago Blue dye solution (Sigma, St. Louis, MO) in saline.
In study 2, female mice (8–10 wk old) were ovariectomized and rested for 10 days. The mice were given an injection of sesame oil (vehicle control [CO]; 0.1 ml/mouse; Sigma), estradiol-17β (E2; 100 ng/mouse; Sigma), P4 (2 mg/mouse; Sigma), or a combination of E2 and P4 (E + P) subcutaneously. Uteri were collected 6 and 24 h after injection using methods described previously .
Uteri were fixed in fresh 4% paraformaldehyde in PBS at room temperature for 8–12 h and embedded in paraffin for in situ hybridization analyses (n = 6) or were snap frozen in liquid nitrogen and stored at −80°C for RNA extraction (n = 6).
Expression of the Wnt and Fzd genes was studied by RT-PCR as described previously [15, 22–25]. Primers were designed from conserved mouse cDNA sequences using Primer 3 . A partial mouse cDNA of 200–600 bp was cloned by RT-PCR using total RNA isolated from pregnant mouse uteri. Primer and annealing temperatures used for PCR are provided in Table 1. The amplified PCR products were subcloned into the pCRII cloning vector using a T/A Cloning Kit (Invitrogen Life Technologies, Carlsbad, CA) and sequenced in both directions using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA) to confirm identity.
In situ hybridization analysis of mouse uteri was conducted using methods described previously . Briefly, deparaffinized, rehydrated, and deproteinated cross-sections (5 μm) of the uterine horns from each mouse were hybridized with radiolabeled sense or antisense cRNA probes generated from linearized plasmid DNA templates using in vitro transcription with [35S-α]UTP. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB liquid photographic emulsion (Kodak, Rochester, NY), stored at 4°C for 4–60 days, and developed in Kodak D-19 developer. Slides were then counterstained with Gill modified hematoxylin (Stat Lab, Lewisville, TX), dehydrated through a graded series of alcohol to citrisolve (Fisher), and protected with a coverslip.
Images of representative fields of sections hybridized with antisense or sense cRNAs were recorded under brightfield or darkfield illumination with a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera using constant image acquisition parameters to ensure accurate comparison.
Total RNA (n = 6) was isolated from mouse uteri using the Trizol reagent (Gibco-BRL, Bethesda, MD) according to the manufacturer's recommendations. The quantity and quality of total RNA were determined by spectrometry and denaturing agarose gel electrophoresis, respectively. The cDNA was synthesized from total RNA (2 μg) using iScript Select cDNA synthesis Kit (Bio-Rad). Polymerase chain reaction analysis of mRNA expression was performed using an MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) with iQ SYBR Green supermix (Bio-Rad) as the detector according to manufacturer's recommendations. Primers were designed to amplify cDNAs of around 100 bp to maximize efficiency and are summarized in Table 2. Polymerase chain reaction cycle parameters were 95°C for 15 sec and 60°C for 1 min for 40 cycles. The threshold line was set in the linear region of the plots above the baseline noise, and threshold cycle (CT) values were determined as the cycle number at which the threshold line crossed the amplification curve. Polymerase chain reaction without template or template substituted with total RNA was used as a negative control to verify experimental results. After amplification, the specificity of the PCR was verified by both melt-curve analysis and gel electrophoresis to verify that only a single product of the correct size was present. Data are shown as the average fold increase, with standard error (SEM) normalized against Gapdh mRNA. The fold changes are equivalent to 2x−y, where x is the CT value of the control (D1 uteri in study 1 or CO uteri in study 2), and y is the CT value of D4 to D7 in study 1 or the treatments of E2, P4, or E + P in study 2.
All quantitative real-time PCR data were subjected to least-squares ANOVA using the General Linear Models procedures of the Statistical Analysis System (SAS Institute, Cary, NC). Real-time PCR data were corrected for differences in sample loading using the Gapdh data as a covariate. Tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P value of 0.05 or less was considered significant vs. implantation and nonimplantation sites in study 1, and each treatment in study 2. Data are presented as least-square means with SEM.
Reverse transcription-PCR was conducted using primers generated from conserved regions of the 19 Wnt and 10 Fzd genes and the total RNA isolated from adult mouse uteri (D1 to D7). Partial cDNAs of the correct predicted size (Table 1) were cloned and sequenced in both directions to confirm identity. The RT-PCR analyses detected expression of 10 of the 19 Wnt genes (Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt10a, Wnt11, and Wnt16) and 8 of the 10 Fzd receptors (Fzd2, Fzd3, Fzd4, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10) in the adult mouse uterus (data not shown). However, in situ hybridization analyses were only able to detect Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd2, Fzd4, and Fzd6 in the mouse uteri. The partial mouse Fzd2 cDNA shared significant identity (95%) with Fzd1. However, Fzd1 mRNA was undetectable in the mouse adult uterus by quantitative real-time RT-PCR. Therefore, the probe from partial Fzd1/2 cDNA used for in situ hybridization analyses likely detected Fzd2 mRNA.
In situ hybridization analysis was used to examine the temporal and cell-specific expression of Wnt and Fzd genes before implantation (D1 and D4), at the onset of implantation (D5), and after implantation during decidualization (D6 and D7; Figs. Figs.11–4).
As illustrated in Figure 1, Wnt4 mRNA was detected in endometrial LE and stroma on D1 and was almost undetectable in the uterus on D4. On D5 (just after the onset of implantation ), Wnt4 mRNA was localized in the stroma surrounding the implanting blastocyst (denoted by the arrowhead in Fig. 1). Wnt4 mRNA became more abundant on D7 as expression expanded to the secondary decidual zone (SDZ) and was approximately 15-fold higher than levels on D1. However, Wnt4 mRNA was not observed in the portion of the uterus lacking implantation sites. Wnt5a mRNA was observed at low abundance in the stroma underlying the LE on D1 and D4. On D5, Wnt5a mRNA was more abundant in the mesometrial pole of stroma adjacent to the LE in the implantation site and restricted to only the mesometrial decidual zone on D6 and D7. The expression of Wnt5a was lower in the nonimplantation site.
Wnt7a mRNA was detected only in the LE, and maximal abundance of Wnt7a mRNA was observed in the LE in the implantation site on D5 and declined thereafter in both implantation and nonimplantation sites (Fig. 2). Wnt7b mRNA was detected primarily in the LE on D1 and D4. Wnt7b mRNA remained abundant, was robust on D5 in the implantation site, and was approximately 15-fold higher than that of D1. At the onset of implantation (D5), Wnt7b mRNA was concentrated at the site of implantation and was 7-fold higher than in the nonimplantation site.
Wnt11 mRNA was very low to undetectable in the uterus on D1 (Fig. 3). The abundance of Wnt11 mRNA increased in the LE and upper endometrial glands on D4 (greater than 10-fold) and decreased in the LE and upper glands on the antimesometrial side prior to implantation between D4 and D5. Wnt11 mRNA disappeared adjacent to the implanting embryo but remained abundant in mesometrial LE on D5 in the implantation site. After D6, Wnt11 was no longer detected in the uteri of pregnant mice. Wnt16 mRNA was observed specifically in the upper stroma of the uterus on D1 and became more abundant on D4. Wnt16 mRNA was localized only in the stroma surrounding the LE and remained in the stroma adjacent to the LE in the implantation site but not in the primary decidual zone (PDZ) on D5. Wnt16 mRNA was lower in abundance in the uteri in the nonimplantation site.
As illustrated in Figure 4, Fzd2 mRNA was extremely low on D1, was detected in the stroma on D4, increased in the endometrial stroma surrounding the implantation site on D5, and expanded to both mesometrial and antimesometrial decidual zones thereafter. No differences in Fzd2 mRNA abundance were found between implantation and nonimplantation sites. Fzd4 mRNA was lower in abundance on D1, then appeared in all major uterine cell types in the LE, GE, and stroma as well as in the vascular endothelial cells on D4. The abundance of Fzd4 mRNA increased in the stroma adjacent to LE on D5 and around the embryo on D6 in the implantation site, and the mRNA was abundant in the mesometrial decidual zone on D7. However, Fzd4 mRNA was not different in implantation or nonimplantation sites. Fzd6 mRNA was abundantly detected in the LE and GE on D1, and it remained lower in the LE and GE on D4 and D5. On D6 and D7, Fzd6 mRNA was localized in the decidua in the implantation site and endometrial epithelia in the nonimplantation site, as well as in vascular endothelial cells in the myometrium. Fzd6 expression was not different between implantation and nonimplantation sites.
The differential patterns of Wnt and Fzd on D1 through D7 pregnancy suggested that these genes are regulated by ovarian estrogen and/or progesterone. Therefore, effects of hormone replacement were examined on expression of these genes in ovariectomized mice using a previously established model . Quantitative real-time PCR was used to determine mRNA levels at both 6 and 24 h after hormone injections, and in situ localization of gene expression at 6 h (Figs. (Figs.55–7) and 24 h (data not shown because the spatial localizations of each mRNA at 6 and 24 h were identical) was performed.
Wnt4 mRNA was low in the uterus of ovariectomized mice receiving corn oil (CO) vehicle as a control (Fig. 5). A single injection of E2 to ovariectomized mice increased Wnt4 mRNA abundance approximately 4.5-fold in the endometrial stroma at 6 h (CO vs. E2; P < 0.05). Administration of P4 alone did not (P > 0.10) affect Wnt4 expression, but it decreased the E2 induction of Wnt4 when coadministered (E2 vs. E2 + P4; P < 0.05). However, by 24 h the effect of a single E2 injection on Wnt4 expression was no longer detected (P > 0.10) in the ovariectomized mouse uterus. Wnt5a mRNA expression was not altered (P > 0.10) by ovarian steroids at either 6 or 24 h (Fig. 5). Wnt7a was detected in the LE in the ovariectomized mice treated with only CO. Although E2 treatment did not alter uterine expression of Wnt7a at 6 h, a single injection of P4 as well as E2 and P4 reduced (P < 0.05) Wnt7a mRNA abundance at 6 h. At 24 h, Wnt7a expression in the ovariectomized mouse uterus was reduced (P < 0.05) by all ovarian steroid treatments (Fig. 5).
Although the Wnt7b mRNA was low in the CO-treated uteri, E2 increased (P < 0.05) Wnt7b in mRNA in the LE at 6 and 24 h by approximately 2- and 3-fold, respectively (Fig. 6). In contrast, P4 had no effect (P > 0.10) on Wnt7b expression. A combined treatment of both hormones stimulated Wnt7b expression at 6 h (P < 0.05) but not at 24 h. Wnt11 was also regulated by ovarian steroid hormones (Fig. 6). Wnt11 mRNA was predominantly detected in the endometrial epithelia in CO-treated uteri. A single injection of P4 increased (P < 0.05) expression of Wnt11 in the LE by about 3-fold at 6 h, whereas E2 treatment decreased (P < 0.05) Wnt11 mRNA abundance by 10-fold. Indeed, Wnt11 mRNA was not detected in the uteri of E2-treated mice. The upregulation of Wnt11 expression by P4 was augmented (~6-fold) in the uteri at 24 h (P < 0.05). A combined treatment also increased (P < 0.05) Wnt11 expression at 6 and 24 h. The expression of Wnt16 was suppressed by ovarian steroid hormones (P < 0.05), although Wnt16 mRNA abundance appeared low in the ovariectomized mouse uterus receiving CO (Fig. 6).
The expression of Fzd receptors (Fzd2, Fzd4, and Fzd6) showed similar patterns from treatments of ovarian steroid hormones (Fig. 7). Although these three receptors were expressed in uteri from ovariectomized animals, treatment with E2 reduced (P < 0.05) the expression of Fzd2, Fzd4, and Fzd6 in the mouse uterus at 6 and 24 h, but P4 treatment had no effect (P > 0.10). The combination of E2 and P4 also decreased (P < 0.05) the expression of these receptors.
Results of the present study indicate that Wnt genes (Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, and Wnt16) and Wnt receptors (Fzd2, Fzd4, and Fzd6) are present in the mouse uterus during the peri-implantation period of pregnancy. Our previous study in sheep found that WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT7A, and WNT11) and their signaling pathways are present in endometria and/or conceptuses during early pregnancy . Tulac et al.  found that WNT genes (WNT2, WNT3, WNT4, WNT5A, WNT7A, and WNT8B) were expressed in human endometria during the secretory phase of the menstrual cycle. In mice, Wnt4 and Wnt5a have been shown previously to be expressed predominantly in uterine stroma during the estrous cycle, whereas Wnt7a is expressed only in the LE . The localization of Wnt genes (Wnt4, Wnt5a, and/or Fzd2) in the adult uterus, including the peri-implantation period, and ovarian steroid hormones regulate their expression in the mouse uterus [17, 30]. The present study revealed that Wnt7b, Wnt11, and Wnt16; Fzd4 and Fzd6 receptors; and Wnt4, Wnt5a, Wnt7a, and Fzd2 were temporally and spatially expressed in the mouse uterus during peri-implantation and regulated by ovarian steroid hormones. Collectively, these studies suggest that Wnt genes are expressed in the adult uteri of mice as functional regulators of implantation and implicate functional WNT systems as conserved regulators of adult uterine function across mammals.
The proteins of the WNT family are highly conserved secreted signaling molecules involved in cell-to-cell interactions during embryogenesis . The majority of the functional information concerning the role of the WNT family in adult uterus comes from the study of Wnt4. Wnt4 is required for stromal decidualization, the establishment of embryo and maternal communication, and the progression of implantation in human and mouse [18, 19]. Wnt4 was identified as a downstream target gene of Bmp2 signaling in stromal decidualization [18, 19]. Bmp2 serves as a molecular integrator of peri-implantation events to coordinately regulate a molecular system necessary for stromal cell differentiation [18, 19, 32]. During implantation, Wnt4 and Bmp2 are strongly induced in the PDZ, with their expression expanding to the SDZ [17, 32]. The present study found that expression of Wnt4 was limited to the implantation sites and was not present in nonimplantation sites. Further, the expression of Wnt4 is abrogated in the conditional ablation of Bmp2 uterus . Thus, Wnt4 is a critical regulator of stromal decidualization linked with Bmp2.
WNT proteins bind to the FZD family of receptors. The classical canonical WNT signaling pathway suggests that after completing several cytoplasmic relay components, the signal is transduced to CTNNB1 (β-catenin), which subsequently enters into the nucleus, forming a complex with TCF/LEF to activate transcription of the Wnt target genes [31, 33]. In mice, the canonical WNT signaling pathway plays a central role in coordinating blastocyst-uterine interactions required for implantation . Mohamed et al.  demonstrated that the activation of nuclear CTNNB1 in response to the implanting embryo occurred during implantation, and WNT7A, through WNT/CTNNB1 signaling, appears to be particularly effective in triggering implantation responses. Moreover, activation of the canonical WNT signaling pathway required the presence of the blastocyst, and inhibition of WNT/CTNNB1 signaling interfered with implantation . Total and active CTNNB1 are highly localized in the PDZ around the antimesometrial pole of the LE, where the implanting blastocyst is located, and in the stroma just outside of the PDZ and adjacent to the LE around the mesometrial part of lumen . In the present study, Wnt7a mRNA was detected in the LE on D1, with the expression remaining strong through D4 and increasing in abundance on D5 in the LE, including the implantation chamber. Thus, these results support that Wnt7a in the LE is a critical factor for blastocyst-uterine interactions to regulate initiation of the decidualization process through canonical WNT/CTNNB1 signaling. Indeed, WNT7A stimulates proliferation of ovine trophectoderm cells through the canonical WNT/CTNNB1 pathway .
It is likely that Wnt7b, which is thought to act via the canonical WNT pathway, regulates implantation. In the present study, the expression of Wnt7b was strongly upregulated on D5 within implantation sites. A single injection of E2 to ovariectomized mice increased Wnt7b expression in the uterus, suggesting that Wnt7b may be directly stimulated by nidatory E2 to regulate embryo-uterine interactions. However, ovarian steroids downregulated the uterine expression of Wnt7a in ovariectomized mice. We did not see any difference in expression of Wnt7a in both implantation and nonimplantation sites of pregnancy. Thus, the respective roles of Wnt7a and Wnt7b could be distinct during implantation. On the other hand, adult Wnt7a-deficient mice are viable but infertile because of the lack of endometrial glands [34, 35]. Wnt7b-null mice die in utero during midgestation because of placental abnormalities involving the fusion of the chorion and allantois . Although the functional role of Wnt7b in the adult mouse is currently unknown, the loss of Wnt7b in the LE may result in a major defect of stromal cell differentiation at the beginning of decidualization prior to the formation of the placenta.
WNTs can also have biological effects via a noncanonical pathway mediated by WNT/Ca2+ and WNT/JNK pathways . It is currently unknown whether the noncanonical WNT pathway is important for uterine biology in mice. Indeed, the actions of Wnt11 and Wnt16 are thought to be solely mediated by the noncanonical pathways. In the present study, we found temporal and cell-specific differences in Wnt11 and Wnt16 expression in the prereceptive and the receptive uterus around the window of implantation. The functional roles of Wnt11 and Wnt16 in the adult mouse uterus are currently unknown, because Wnt16-null mice have not yet been generated, and characterization of Wnt11-null mice is impossible because of perinatal lethality . The expression levels of Wnt11 and Wnt16 were quite low or almost undetectable on D1, suggesting that preovulatory estrogen and/or ovulatory hormones do not regulate these Wnt genes in the uterus. On D4, the expression of Wnt11 and Wnt16 increased and remained high on D5. A single injection of P4, as well as a combination of E2 and P4, to ovariectomized mice upregulated the expression of Wnt11 in the endometrial epithelia. Thus, the increase in Wnt11 is probably regulated by increasing levels of P4 from the corpora lutea, although the upregulation of Wnt16 remains unclear because of its not being directly stimulated by P4 in the ovariectomized uterus.
The process of implantation consists of three stages—apposition, attachment, and invasion of the uterine LE [1, 2]. The apposition of the blastocyst to the LE, which is the first step in the implantation process, is initiated by the creation of an implantation chamber surrounding each blastocyst along the uterine lumen. This is followed by the attachment of the blastocyst trophectoderm to the LE at the antimesometrial pole of the uterus. This attachment leads to invasion of the embryo through the LE and basal membrane into stroma and extensive proliferation and differentiation of stromal cells to decidual cells to establish a vascular relationship with the mother [1, 2, 6]. Wnt11 was expressed in the LE and then disappeared in the LE surrounding the implanting blastocyst on the antimesometrial side of the uterus on D5. We found that the expression of Wnt11 was quite low in the GE, whereas Wnt11 is abundant in the GE in the uterus of early pregnant sheep . Wnt16 was highly expressed in the stroma adjacent to the LE on D4 and disappeared in the stroma surrounding the PDZ on D5. Of particular note, Wnt16 is one of the upregulated Foxo1-dependent transcriptional targets involved in the human decidualizing cell cross-talk with progesterone receptor (PGR) . On the other hand, noncanonical WNT signaling is one of the key pathways to regulate cell polarization and movement , including orientation of cilia  and sensory hair cells . Thus, it is tempting to speculate that the fading expression of both Wnt11 and Wnt16 around the implanting blastocyst may be involved in orientating the implantation chamber and specifies blastocyst attachment at the time of implantation, possibly through interaction with LIF, a cytokine known to be a critical factor for implantation . Interestingly, Wnt11 mRNA was only detected in embryos that undergo the morula-to-blastocyst transition during early pregnancy but not upregulated when blastocysts are trapped in the oviduct following ligation of the uterotubal junction .
The function of Wnt5a is yet to be established in adult mice, but in ewes it stimulates conceptus trophectoderm migration via the noncanonical WNT signaling pathway [15, 44, 45]. WNT5A regulates human endothelial cell proliferation and migration to promote angiogenesis and is associated with tumor proliferation . In the present study, the expression of Wnt5a was abundant in the mesometrial decidual cells around the mesometrial region during decidualization. Cells in the mesometrial region are the last to initiate decidualization, forming the mesometrial decidua, the area for placental vascular supply. Thus, Wnt5a may be a factor to stimulate proliferation in decidual and/or endothelial cells in the mesometrial decidua to regulate placental development, probably to support conceptus growth. On the other hand, the expression of Wnt5a during the window of prereceptive and receptive uterus does not appear to be under the influence of ovarian steroids. We observed very low Wnt5a expression before D5 during peri-implantation, and a single treatment of ovarian steroids did not restore expression in ovariectomized animals. However, a previous study  found that the expression of Wnt5a in ovariectomized mice was weakly regulated by estrogen. The regulation of Wnt5a expression by ovarian steroids thus remains unclear.
To be effective in autocrine and/or paracrine signaling, WNT proteins must associate with their extracellular surface receptors frizzled to mediate intracellular signal transduction pathways . It has been shown that Fzd2 is present in the mouse uterus , FZD4 is involved in retinal angiogenesis , and FZD6 was observed in the endometrial epithelia during early pregnancy in sheep . In the present study, Fzd2 was expressed in stromal cells, and its expression was expanded to stromal decidual zone. Fzd4 appeared to be in blood vessels as well as in the stroma surrounding the embryo during decidualization, and Fzd6 was detected in the endometrial epithelia and stroma, and it appeared to be in some vessels in the myometrium. Although the specific receptor-ligand binding relationships have yet to be established in the uterus, all three Wnt gene receptors we examined are expressed in nearly all of the adult uterine cells during implantation. Thus, we hypothesize that Wnt genes that are expressed in the adult mouse uterus mediate intracellular signal transduction via their receptors and play critical roles for implantation, including stromal decidualization.
The events of embryo implantation that are necessary for a successful pregnancy are tightly regulated by steroid hormones, E2 and P4, which act through their receptors, estrogen receptor (ESR) and PGR [1, 2, 48]. Many mediators and effectors of ESR and PGR have been investigated in the critical implantation stages in specific cell types of the mouse uterus [1, 2, 48]. Although we do not know conclusively the relationships and interactions of Wnt genes and ovarian steroid signaling, results of the present studies indicate that multiple Wnt genes are expressed in the adult mouse uterus and are candidates to regulate implantation. In summary, the findings of the present study provide a foundation for future experiments directed at uncovering the functional role of each WNT and its signaling pathway during implantation.
1Supported in part by National Institutes of Health (NIH) grant 5 R21 HD054679 to T.E.S., and NIH grant P30 ES09106 and Southern Illinois University School of Medicine CRC, FAS no. 4-14124 to K.H.