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Departments of Pediatrics (T.D., H.S., Y.G., S.K.Da., S.K.De.), Cell and Developmental Biology and Pharmacology (S.K.De.), Vanderbilt University Medical Center, Nashville, Tennessee 37232; and Target Discovery and Department of Pharmacology (A.R., S.M.), NV Organon, 5340 BH Oss, The Netherlands
Address all correspondence and requests for reprints to: Sudhansu K. Dey, Vanderbilt University Medical Center, Developmental Biology, MCN-D4100, Nashville, Tennessee 37232-2678. E-mail: sk.dey{at}vanderbilt.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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LIF is a member of the four-helix-bundle cytokines belonging to the IL-6 family that includes IL-6 itself, oncostatin M, ciliary neurotrophic factor, cardiotrophin, and others (1, 2, 3, 4, 5, 9). LIF binds to its ligand-specific receptor and uses gp130 as a shared signal-transducing receptor partner (5, 9). Engagement of gp130 results in the activation JAK/Tyk tyrosine kinases and signal transducer and activator of transcription family of transcription factors, STAT (9). Inactivation of gp130 by deleting all signal transducer and activator of transcription binding sites results in implantation failure (10), further suggesting the importance of LIF signaling in implantation. Uterine LIF is also implicated in implantation in various other species including humans (11, 12, 13, 14, 15).
Previously, LIF was shown to be transiently expressed in mouse uterine glands on d 4 of pregnancy, suggesting its role in uterine preparation for implantation (16). However, our recent studies show that uterine Lif expression is biphasic on d 4 of pregnancy; not only is Lif expressed in glands, but also in stromal cells surrounding the blastocyst at the time of the attachment reaction (8). This suggests that LIF has dual roles: first in the preparation of the uterus and later in the attachment reaction. However, the nature of the complex ligand-receptor interaction and detailed expression patterns of the LIF ligand-specific receptor and gp130 in the uterus during implantation still remain largely unanswered and so is the molecular mechanism by which LIF executes its effects on implantation.
Hox genes are developmentally regulated transcription factors belonging to a multigene family. They share a common highly conserved sequence element called the homeobox, which encodes a 61-amino acid helix-turn-helix DNA-binding domain (17). Several Hox genes at the 5'-end of each cluster are classified as abdominalB (AbdB)-like Hox genes because of their homology with the Drosophila AbdB gene. Hoxa-10 is an AbdB-like Hox gene that is expressed in developing genitourinary tract during development and in the adult uterus during pregnancy (18). Hoxa-10 mutant mice exhibit oviductal transformation of the proximal one third of the uterus and show implantation and decidualization failures. Implantation failure is unrelated to oviductal transformation (18) but is attributed to reduced uterine stromal cell proliferation in response to progesterone (18, 19). Hoxa-10 is markedly up-regulated in the uterus during the mid-secretory phase in a steroid hormone-dependent manner, suggesting their roles in human implantation (20). Hoxa-10 is implicated in the local events of cellular proliferation by regulating cell cycle molecules. Indeed, while cyclin D3 is aberrantly expressed in uteri of Hoxa-10 null mice (21), cyclin-dependent kinase inhibitors p15 and p57 are up-regulated in uteri of these mice (22).
Although several members of the epidermal growth factor (EGF) family of growth factors and cyclooxygenase-2 are aberrantly expressed in uteri of Lif mutant mice at the anticipated time of implantation, uterine expression of several others genes including Hoxa-10, steroid hormone receptors and proangiogenic factors are normal in uteri of these mice during the preparatory phase (8). Similarly, whereas genes associated with prostaglandin signaling and cell cycle regulation are aberrantly expressed in Hoxa-10 mutant uteri, the expression of Lif, Hoxa-11, and several other genes are normally expressed (19). These results suggest that LIF signaling during implantation is independent of Hoxa-10 signaling.
Because homeotic and Wnt signaling are crucial to early developmental processes and considered important for uterine biology (17, 18, 23, 24, 25), we compared the status of another homeobox gene Msx-1, and of Wnt4 and its antagonist sFRP4 in wild-type uteri with that in uteri missing the Lif or Hoxa-10 gene during the periimplantation period and under steroid hormonal stimulation. Our objective was to see whether a molecular network involving cytokine, homeobox, and Wnt signaling is operative in the uterus for implantation. We report here that Msx-1, Wnt4, and sFRP4 are differentially regulated in the wild-type uterus in a spatiotemporal manner during implantation. We also show that, whereas both Msx-1 and Wnt4 signaling become aberrant in Lif mutant uteri, primarily Wnt4 signaling is disrupted in Hoxa-10 mice, delineating a novel but complex networking among cytokine, homeotic, and Wnt signaling in implantation.
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RESULTS |
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We first examined the Wnt4 and Msx-1 signaling in the uterus at different phases of the periimplantation period (0900 h on d 1, 4, 5, and 8). We selected to examine Wnt4 expression in parallel with sFRP4 to better understand Wnt4 signaling in the uterus. We have previously shown that Wnt4 is expressed in stromal cells surrounding the blastocyst with the onset of implantation followed by its expression in the deciduum with the progression of pregnancy (27). As shown in Fig. 1A
, Wnt4 and sFRP4 show unique expression patterns in the uterus during the periimplantation period. Low levels of Wnt4 were expressed in the luminal epithelium on d 1, whereas the expression was undetectable in any major uterine cell types on d 4 of pregnancy. On d 5, Wnt4 expression was localized to the stromal cells immediately surrounding the implanting blastocyst. The expression became more intense thereafter, and on d 8 the expression expanded to the secondary decidual zone. In contrast to Wnt4 expression, sFRP4 showed a different expression pattern in the uterus during the periimplantation period (Fig. 1A). We observed a modest level of expression of sFRP4 that was limited to the connective tissue in the myometrial bed on d 1 of pregnancy. On d 4, signals were present in a select population of stromal cells throughout the endometrium and in stromal cells just underneath the myometrium. After implantation on d 5 and thereafter, sFRP4 expression became more intense and was primarily restricted to the undifferentiated stromal cells forming a dividing zone between the circular muscle layer and the deciduum. The expression was also present in the circular muscle layer and in connective tissues between the circular and longitudinal muscle layers. Collectively, the results suggest that the process of implantation differentially regulates the expression of Wnt4 and sFRP4. The results of Northern blot hybridization performed on total RNA samples of whole uteri are more or less consistent with the in situ hybridization results (Fig. 1B). For examples, two transcripts (
2 and 4 kb) for Wnt4 mRNA were detected for uterine RNA and the levels were higher on d 8 than other days examined. With respect to sFRP4 expression, again two transcripts (2 and 3.5 kb) were detected and the levels reflected in situ results when corrected for rPL7 (a housekeeping gene) expression. However, it is to be recognized that Northern blot analysis alone using whole uterine RNA samples does not provide information on cell type-specific expression, and the dilution effects resulting from heterogeneous uterine cell types in which myometrial cells contribute for most of the isolated RNA may compromise meaningful interpretation of the results if not combined with in situ hybridization experiments.
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). It is undetectable in the uterus on d 1 of pregnancy, but high levels of expression were noted in the luminal and glandular epithelia on d 4 of pregnancy. The expression was down-regulated with the onset of implantation on d 5 and became undetectable with the progression of implantation on d 8. Results of Northern hybridization of uterine RNA samples harvested before, during and after implantation shows a good correlation with our in situ hybridization results (Fig. 2, A and B). Furthermore, higher uterine expression that is present on d 4 of pregnancy or pseudopregnancy showed down-regulation with the initiation of implantation on d 4 night (Fig. 2C) and ultimately disappeared with the progression of implantation (Fig. 2, A and B). Collectively, the results suggest that up-regulation of Msx-1 is important for the uterus to become receptive, whereas its down-regulation is required for the initiation and progression of implantation.
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, although Wnt4 is undetectable in d 4 pregnant uteri, it is expressed in stromal cells at the site of blastocyst with the onset of implantation on d 5 in wild-type mice. In contrast, sFRP4 is expressed in stromal cells on d 4 and becomes more defined and prominent with the onset of implantation as observed on d 5. Thus, we wanted to compare the expression pattern of sFRP4 in wild-type and Lif(–/–) uteri at 0900 h and 1800 h on d 4 of pregnancy. The morning and evening time points were chosen to explore the differential importance of LIF in uterine gene expression with respect to uterine preparation and blastocyst attachment for implantation. This rationale is based on our previous observation of biphasic uterine expression of Lif on d 4 of pregnancy and molecular interaction between the implantation-competent blastocyst and uterine luminal epithelium on the evening of d 4 (8, 26). We observed that sFRP4 expression at both time points on d 4 was similar in wild-type uteri. However, in Lif mutant mice, the uterine expression was dramatically down-regulated in all cell populations that showed higher expression in wild-type uteri (Fig. 3A). These results suggest that a Wnt signaling component becomes aberrant in the absence of LIF. We next asked whether Hoxa-10 influences Wnt4 signaling.
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). These results suggest that Wnt4 signaling becomes aberrant in uteri of mice missing Hoxa-10. This aberrant Wnt4 signaling may be one cause of severely compromised decidualization leading to infertility in these mice. Although we cannot rule out the possibility that aberrant expression of Wnt4 and sFRP4 on d 5 is secondary to altered cellular changes in the uterus lacking Hoxa-10, it is to be recalled that implantation failure in most Hoxa-10 mutant females is primarily due to defective decidualization after the initial attachment reaction (18).
Msx-1 Is Aberrantly Expressed in the Uterus with the Loss of LIF Functions
Our results as described above in wild-type mice (Fig. 2) suggested that down-regulation of Msx-1 is influenced by the implanting blastocyst. To examine whether Msx-1 expression is indeed regulated by a signal originating from an implanting blastocyst, we first compared the expression profiles between wild-type and Lif mutant mice on d 4 of pregnancy during the uterine preparatory (0900 h) and preattachment (1800 h) phases of the implantation process by in situ hybridization (Fig. 4A). We observed that Msx-1 that is expressed, as expected, in the uterine epithelium on d 4 mornings is down-regulated in the afternoon in wild-type mice. In Lif mutant mice, whereas the uterine expression of Msx-1 on d 4 morning is similar to that of wild-type mice, its down-regulation is not evident in the evening of d 4 (Fig. 4A). To gain further insight regarding the regulation of Msx-1 during implantation, we compared its uterine expression pattern between pseudopregnant and pregnant wild-type and Lif mutant mice on the mornings of d 4 and 6 (Fig. 4B). Similar to our earlier observation, Msx-1 was expressed in the luminal and glandular epithelia on d 4 mornings at comparable levels in both wild-type and Lif mutant mice. Surprisingly, Msx-1 expression was dramatically down-regulated in uterine epithelia of both pseudopregnant and pregnant wild-type mice on d 6, but the expression persisted in the epithelium of Lif mutant uteri under similar conditions. These results suggest that implanting blastocyst is not the primary cause for the down-regulation of Msx-1, but one or more other factors is responsible for this down-regulation after d 4 of pseudopregnancy or pregnancy. This would then also suggest that one or more such factors is not operative in Lif mutant mice to down-regulate Msx-1 expression. Alternatively, the changing levels of steroid hormones perhaps influence this gene during pregnancy or pseudopregnancy and that the uterus loses this responsiveness in the absence of LIF.
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). This suggests that epithelial expression of Msx-1 is independent of the regulation by stromal cell Hoxa-10.
Ovarian Steroid Hormones Differentially Regulate sFRP4 and Msx-1 Expression in the Uterus
Our observation of temporal and cell type-specific uterine expression of differentially expressed sFRP4 and Msx-1 on d 1 and 4 of pregnancy suggests that these genes are regulated by ovarian estrogen and/or progesterone. Therefore, we further examined the expression of these genes in a more defined system, i.e. in ovariectomized uteri after steroid hormone treatments.
Although sFRP4 showed temporal and cell-specific expression during the periimplantation period, regulation of this gene by ovarian steroids was not robust (Fig. 5, A–D). The results of in situ hybridization show that the expression persists in ovariectomized uteri of both wild-type and Lif mutant mice and is little altered by progesterone treatment at 6 or 24 h. However, a single injection of estrogen down-regulated the expression of sFRP4 at 6 h, but not at 24 h. A combined treatment of progesterone and estrogen also showed somewhat reduced expression of sFRP4 at 24 h (Fig. 5, A and B). The results of Northern hybridization of sFRP4 when corrected for rPL7 also suggest that the steroid effects on uterine sFRP4 regulation are very modest, if any (Fig. 5, C and D). Thus, the robust expression of this gene with the onset of implantation is not likely due to the interplay of progesterone and/or estrogen; rather, one or more factors other than these steroids or in combination with these steroids is responsible for regulating this gene in the uterus during implantation and is inoperative in Lif mutant uteri. Although not examined, the expression of Wnt4 in the uterus does not appear to be under the influence of ovarian steroids because its expression is very low on d 1 and undetectable on d 4 of pregnancy.
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, A–D) also appears to be modestly regulated by ovarian steroids. As assessed by in situ hybridization, the expression was present in the luminal and glandular epithelia in wild-type uteri after ovariectomy, whereas the signal intensity was somewhat higher in ovariectomized Lif mutant uteri at 6 h after oil (control) injection. Progesterone treatment slightly up-regulated the expression in these cell types primarily in wild-type, but not in mutant, mice at 6 h. Estrogen treatment also did not alter uterine expression of Msx-1 either in the wild-type or Lif mutant ovariectomized mice at 6 h as compared with oil treatment (Fig. 6A). The uterine expression of Msx-1 persisted 24 h after an oil injection in both wild-type and mutant mice. However, although estrogen treatment down-regulated the expression, progesterone modestly up-regulated this expression in ovariectomized mice at 24 h irrespective of the genotypes. The expression persisted in both the wild-type and Lif mutant females when progesterone treatment was combined with that of estrogen (Fig. 6B). Northern blot results based on the ratio of band intensities between Msx-1 and rPL7 mRNAs also suggest little effects of steroids on uterine Msx-1 expression at 6 or 24 h in ovariectomized mice (Fig. 6, C and D). Collectively, the results suggest that regulation of Msx-1 in the uterus is complex. Although the absence of LIF favors up-regulation of Msx-1 expression during pregnancy or pseudopregnancy, this was not obvious in ovariectomized mice after steroid hormone treatments, suggesting other factors in addition to ovarian steroids are important for uterine regulation of Msx-1 in intact mice.
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DISCUSSION |
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The proteins of the Wnt family are highly conserved secreted signaling molecules involved in cell-to-cell interactions during embryogenesis (30, 31, 32). Wnt signaling is also indicated in tumorigenesis (33). Wnt proteins bind to the Frizzled (Fzd) family of receptors that are displayed on the cell surface. The classical canonical pathway suggests that the signal after completing several cytoplasmic relay components is transduced to ß-catenin, which subsequently enters into the nucleus forming a complex with TCF to activate transcription of the Wnt target genes (30). However, Wnt proteins also signal through noncanonical pathways that includes Wnt/Ca2+ and Wnt/Jun N-terminal kinase pathways (34). Until now, 19 Wnt and 8 Fzd proteins have been identified in the mouse [Nusse, R., the Wnt gene home page (http://www.stanford.edu/
rnusse/wntwindow.html)], suggesting distinct and overlapping functions of Wnt signaling. Targeted deletion of several Wnt and Fzd genes in mice leads to specific developmental defects [Refs.23 and 24 ; and Nusse, R., the Wnt gene home page (http://www.stanford.edu/rnusse/wntwindow.html)].
Fzd proteins resemble G protein-coupled receptors with a serpentine structure containing seven-transmembrane helical domains and a cytoplasmic carboxy terminal. They are endowed with cysteine-rich domain, known as Fzd domain (31). Several proteins have strong homology with the cysteine-rich domain of Fzd proteins, but lack the transmembrane domain. These molecules are now termed as secreted Fzd-related proteins (sFRPs). Five sFRPs (sFRPs 1–5) have been identified in the mouse. Evidence suggests that sFRPs inhibit Wnt signaling by competing with Fzds for Wnt ligands or in a dominant-negative fashion by forming a nonsignaling complex with Fzds (30). One emerging theme is that sFRPs, acting as anti-Wnts, participate in the regulation of morphogenetic gradients or zones of Wnt signaling. There is also evidence that similar gradient zones created for bone morphogenetic protein (BMP) signaling by its antagonists Noggin and Chordin collaborates with Wnt gradients in specifying cell fates and demarcating tissue morphogens and boundaries, giving rise to the organization of the vertebrate body plan during development (24, 30).
Although the necessity for Wnt and BMP signaling during development is well documented, their roles in maintaining adult tissue morphogens and boundaries remain largely unknown. In mice, Wnt-7a signaling is crucial for the female reproductive tract development along the anteroposterior axis during the postnatal development. It is expressed in the uterine gland and luminal epithelium in newborn mice, and in the luminal epithelium in adult females (23). Targeted deletion of the Wnt-7a gene in mice has shown that cellular characteristics of the oviduct, uterus, cervix, and vagina become aberrant along the anteroposterior axis resulting in global posterior shifting of the reproductive tract with the loss of Hoxa-10 and Hoxa-11 expression. In addition, the uterus lacks glands and the myometrium becomes disorganized in these mutant females. In this context, Hoxa-11 has been shown to regulate uterine stromal cell proliferation and apoptosis during neonatal development (25). These findings provide evidence that Wnt-7a expressed in the epithelium is crucial for maintaining the molecular and morphological boundaries of specific cell populations along the anteroposterior and radial axes of the female reproductive tract (23), whereas Hoxa-11 is required for neonatal uterine development (25). As described in the present investigation, an aberrant uterine expression of sFRP4 and Wnt4 in Hoxa-10 mutant mice suggests that an interaction between Wnt and homeobox signaling is crucial to the implantation process. There is also genetic evidence that Wnt2 is critical to normal placental development (35).
The adult uterus undergoes dynamic cellular and molecular changes during pregnancy, but how these changes are coordinated to specify allocations of new cell types and their boundaries and how the uterus is returned to its original state after successful end of pregnancy are not known. The first step in the implantation process, the apposition of the blastocyst to the luminal epithelium, 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 with the luminal epithelium at the antimesometrial pole of the uterus. This attachment leads to extensive proliferation and differentiation of stromal cells to decidual cells (decidualization) at the site of the implanting blastocyst with luminal epithelial cell apoptosis at the attachment site (6, 36). The decidualization process is initiated at the antimesometrial pole, which then spreads to the mesometrial pole (the presumptive site of placentation), orienting the shape of the uterus in an antimesometrial-mesometrial direction. In mice, the differentiating stromal cells surrounding the blastocyst initially form the primary decidual zone (PDZ) on d 5. The PDZ is avascular and densely packed with decidual cells. By d 6, the secondary decidual zone (SDZ) is formed around the PDZ. At this time, cell proliferation ceases in the PDZ, but still continues in the SDZ (36). However, a thin layer of undifferentiated stromal cells establishes a boundary between the myometrium and SDZ. The PDZ progressively degenerates up to d 8. After d 8, the placental and embryonic growth gradually replaces the SDZ, which is reduced to a thin layer of cells called the decidua capsularis. The mesometrial decidual cells ultimately form the decidua basalis (36). It is still elusive how the implantation chamber is oriented and grows in an antimesometrial-mesometrial direction with decidual reaction spreading in the same direction. It is also unknown how the decidual cell growth is restricted leaving a layer of undifferentiated stromal cells underneath the myometrium.
We speculate that Wnt4 signaling in collaboration with those of BMP and fibroblast growth factor (FGF) helps in orienting the implantation chamber in the antimesometrial-mesometrial direction and specifies these boundaries during implantation and decidualization. Our previous observations of unique cell-specific expression patterns of BMPs and FGFs in the uterus during implantation lend support to this proposition (27). The expression of Wnt4 first in the decidualizing stromal cells surrounding the implanting blastocyst on d 5 and then spreading into cells forming the SDZ on d 8 in parallel with the expression of sFRP4 in a thin layer of undifferentiated stromal cells separating the myometrium from the SDZ suggests that differential Wnt4 signaling participates in establishing this boundary. The expression of Fzd2 follows similar pattern as that of Wnt4 during implantation (Das, S.K., unpublished data). The expression pattern of Wnt4 correlates with stromal cell proliferation during decidualization (36), suggesting its involvement in cell proliferation, a known characteristic of Wnt signaling. We have previously shown an inverse relationship with respect to the expression of BMP2 and its antagonist Noggin during implantation and decidualization (27), again suggesting differential BMP signaling in the uterus during early pregnancy. Furthermore, antimesometrial expression of FGF2 in contrast to that of FGF10 at the mesometrial pole (27) adds evidence that antimesometrial-mesometrial orientation of the uterus during early pregnancy is associated with differential gene expression. We speculate that this uterine orientation also helps in the establishment of the embryonic orientation during development and thus failure of the implantation chamber to orient itself in an antimesometrial-mesometrial direction is likely to disturb embryonic orientation. Although these developmental genes are well known to play crucial roles in establishing boundaries and polarities during embryogenesis, our present results suggest that they are also important for establishing the orientation of the growing implantation chamber and creating boundaries to prevent undifferentiated stromal cells from decidualization and restoring these undifferentiated precursor stromal cells for providing normal stromal tissue after the pregnancy is terminated.
Mouse Msx-1, an ortholog of the Drosophila msh, is involved in several developmental processes (37, 38, 39). It is thought that the regulation of Msx-1 depends on coordinated interactions between the epithelium and mesenchyme involving BMP and Wnt signaling (38). Although Hox genes have critical roles during embryogenesis, their functions during the adult life are limited due to the reduced or lack of developmental plasticity of most tissues. One of the exceptions is the female reproductive tract that is rudimentary at birth but undergoes extensive morphological and functional changes during the reproductive cycles and pregnancy in adult life. Because of this developmental plasticity, it is expected that developmental genes encoding the members of the homeobox, Wnt, and BMP families should have temporal and cell-specific functions in the uterus during pregnancy when the uterus undergoes extensive morphological and functional changes. Msx-1 was shown to be expressed in uteri of nonpregnant mice but drastically down-regulated during pregnancy (37). However, our present study shows that Msx-1 is also expressed during pregnancy but in a temporal and cell-specific fashion with respect to implantation.
Although the expression of Msx-1 in the uterine epithelium on the morning of d 4 is coincident with epithelial differentiation for the preparation of implantation, its down-regulation in the evening of d 4 with approaching implantation and further decreases with the initiation and progression of implantation suggest that a tightly regulated transient expression of Msx-1 is critical to both uterine receptivity and implantation. This is consistent with our present finding of elevated expression of Msx-1 in the evening of d 4 and past the anticipated time of implantation in Lif mutant mice with implantation failure. In contrast to Msx-1 expression in the epithelium, Hoxa-10 and Hoxa-11, another class of homeotic genes, are expressed in uterine stromal cells before implantation and then in decidual cells after implantation. Gene targeting experiments have established that these genes are crucial to implantation and decidualization in mice (18, 19, 40). However, Hoxa-10 is correctly expressed in the uterus lacking LIF (8). This observation together with our present finding of normal expression of Msx-1 in uteri of Hoxa-10 mutant mice suggests that Msx-1 expression is independent of Hoxa-10, but its responsiveness to LIF is unique to the uterus. The definitive role of Msx-1 in uterine biology and implantation will require cell-specific conditional knockout in the uterus because genome wide mutation of this gene produces homozygous null pups that die immediately after birth due to craniofacial defects (39). Nonetheless, our combined results suggest that, whereas a tightly regulated transient expression of Msx-1 in the uterus is important for uterine receptivity and implantation, a coordinated Wnt4 signaling in collaboration with Hoxa-10 signaling is important for maintaining boundaries of the uterine tissue compartments that undergo extensive remodeling during implantation and for establishing the antimesometrial-mesometrial orientation of the implantation chamber.
The down-regulation of Msx-1 in both pregnant and pseudopregnant wild-type mice at the anticipated time of implantation suggests that the implanting blastocyst is not the major mediator of down-regulation of Msx-1. Perhaps progesterone is one of the main inducers of this gene on d 4 mornings, but its down-regulation that occurs in the evening of d 4 and thereafter requires a burst of estrogen. Because this regulation is not observed in pregnant mice missing the Lif gene, one cause of implantation failure in Lif mutant mice could be due to sustained level of Msx-1 in the luminal epithelium. Although it has been suggested that BMP and Wnt signaling are involved in inducing Msx-1 in other tissues during development (38), the expression of BMPs 2, 4, 5, 6, and 8a and b are not detectable in any major uterine cell types on d 4 mornings when Msx-1 is expressed in the uterine epithelium, although low levels of BMP7 transcripts are present in the stroma on d 4. Similarly, as described here (Fig. 1A) and previously reported by us (27), Wnt4 expression is undetectable in the uterus on d 4 mornings. Whether other members of the Wnt family members are expressed in the uterus at this time is not known, although our preliminary results show that Wnt5a, but not Wnt1 or Wnt3, is expressed at low levels in uterine stromal cells on d 4 (Das, S. K., unpublished data).
Another striking observation was that genes that are regulated in an implantation-specific manner are not always regulated by estrogen and progesterone that are essential for uterine preparation for implantation. For example, sFRP4 showed temporal and cell-specific expression in the uterus during implantation, but the responsiveness of this gene to steroid hormonal regulation both in wild-type and Lif mutant mice was insignificant except for its transient down-regulation by estrogen. This would suggest that there are other regulatory factors that modulate uterine gene functions associated with implantation. The reduced uterine expression of sFRP4 in d 4 in the absence of LIF suggests that sFRP4 is important for uterine preparation. Similarly, the regulation of Msx-1 expression by steroid hormones was not robust, except its down-regulation by estrogen at 24 h. Although estrogen alone is capable of down-regulating the expression of Msx-1 at 24 h, its persistent expression in the presence of both estrogen and progesterone indicates that estrogen is less effective in this response in the presence of progesterone. It is possible that down-regulation of Msx-1 during implantation requires other factors in addition to progesterone and estrogen. Persistent expression of Msx-1 in uteri of d 6 pregnant or pseudopregnant Lif mutant mice suggests that LIF is essential for its down-regulation. However, little differential expression of Msx-1 in response to steroid hormones between the wild-type and Lif mutant mice suggests that these steroidal responses in ovariectomized mice appears to be independent of LIF function. Collectively, our results showing down-regulation of sFRP4 and up-regulation of Msx-1 in pregnant Lif mutant mice would suggest that LIF is one of the factors that collaborate with steroid hormones and/or other factors during pregnancy in regulating these genes differentially in the uterus. In conclusion, the present investigation illustrates the importance of a cytokine-homeobox-Wnt signaling network in implantation and pregnancy establishment.
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Hybridization and Analysis
Total and Poly(A)+ RNA Isolation
Total RNA from the Lif mutant or wild-type uteri collected at different times on various days of pregnancy or pseudopregnancy was extracted using TRIzol (Life Technologies, Inc., Gaithersburg, MD). Tissues were homogenized in TRIzol reagent (75 mg tissue/ml) followed by the addition of 0.15 vol of chloroform and vigorous shaking for 15 sec. After incubation for 2.5 min at room temperature, the tubes were centrifuged for 15 min at 12,000 rpm at 4 C. The aqueous phase was precipitated with an equal volume of 2-propanol, stored on ice for 10 min and centrifuged for 10 min at 12,000 rpm at 4 C. The pellet was washed with 75% ethanol and dissolved in diethylpyrocarbonate-treated H2O. The samples were kept on ice for 15 min and subsequently incubated for 10 min at 60 C. RNA quality was checked by agarose gel electrophoresis and RT-PCR. Poly(A)+ RNA was isolated using the oligotex mRNA spin-column protocol (QIAGEN, Valencia, CA; catalog no. 70042). Concentrations of RNA were measured according to the RiboGreen protocol and RNA quality was checked using the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Hybridization Probes
The cDNA clones for the Msx-1, sFRP4, and Wnt4 were generated by RT-PCR cloning with specific primers. For in situ hybridization, sense and antisense 35S-labeled cRNA probes were generated using Sp6 and T7 polymerases, respectively. For Northern hybridization, antisense 32P-labeled cRNA probes for Msx-1, sFRP4, Wnt4, and rPL7 (a housekeeping gene) were generated. Probes had specific activities of about 2 x 109 dpm/µg.
In Situ Hybridization
In situ hybridization was performed as previously described by us (26). In brief, frozen section (10 µm) were mounted onto poly-L-lysine-coated slides and fixed in cold 4% paraformaldehyde in PBS. The sections were prehybridized and hybridized at 45 C for 4 h in 50% formamide hybridization buffer containing the 35S-labeled antisense or sense cRNA probes. Ribonuclease A-resistant hybrids were detected by autoradiography. Sections were poststained with eosin and hematoxylin. Sections hybridized with the sense probes did not exhibit any positive signals and served as negative controls.
Northern Blot Hybridization
For Northern hybridization, total (6 µg) or poly(A)+ RNA (2.0 µg) was denatured and separated by formaldehyde/agarose gel electrophoresis, transferred to nylon membranes, and UV cross-linked. Blots were prehybridized, hybridized, and washed as previously described by us (43).
Treatment of Wild-Type and Lif Mutant Mice with Estrogen and/or Progesterone
To determine whether gene expression responds to estrogen or progesterone in wild-type and Lif(–/–) uteri, mice were ovariectomized irrespective of the estrous cycle and rested for at least 2 wk. They were given an injection of estradiol-17ß (100 ng/mouse) or progesterone (2 mg/mouse). The control mice received sesame oil (0.1 ml/mouse) alone. They were killed at 6 and 24 h after oil or steroid injections, and their uteri were collected for in situ hybridization and Northern hybridization. Steroids were dissolved in sesame oil and injected sc.
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FOOTNOTES |
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This work was supported in parts by grants from the National Institutes of Health (HD12304 and HD33994 to S.K.De., and HD37830 and ES07814 to S.K.Da.).
Abbreviations: AbdB, AbdominalB; BMP, bone morphogenetic protein; EGF, epidermal growth factor; FGF, fibroblast growth factor; Fzd, frizzled; LIF, leukemia inhibitory factor; PDZ, primary decidual zone; SDZ, secondary decidual zone; sFRPs, secreted Fzd-related proteins.
Received for publication October 15, 2003. Accepted for publication February 9, 2004.
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-receptor/gp130 complex. Science 300:2101–2104This article has been cited by other articles:
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  S. K Das Cell cycle regulatory control for uterine stromal cell decidualization in implantation Reproduction, June 1, 2009; 137(6): 889 - 899. [Abstract] [Full Text] [PDF]
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 G. S. Daftary and H. S. Taylor Endocrine Regulation of HOX Genes Endocr. Rev., June 1, 2006; 27(4): 331 - 355. [Abstract] [Full Text] [PDF]
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 F. A. Simmen and R. C. M. Simmen Orchestrating the Menstrual Cycle: Discerning the Music from the Noise. Endocrinology, March 1, 2006; 147(3): 1094 - 1096. [Full Text] [PDF]
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H. N. Jabbour, R. W. Kelly, H. M. Fraser, and H. O. D. Critchley Endocrine Regulation of Menstruation Endocr. Rev., February 1, 2006; 27(1): 17 - 46. [Abstract] [Full Text] [PDF]
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S. J Kimber Leukaemia inhibitory factor in implantation and uterine biology Reproduction, August 1, 2005; 130(2): 131 - 145. [Abstract] [Full Text] [PDF]
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 O. A. Mohamed, M. Jonnaert, C. Labelle-Dumais, K. Kuroda, H. J. Clarke, and D. Dufort From The Cover: Uterine Wnt/{beta}-catenin signaling is required for implantation PNAS, June 14, 2005; 102(24): 8579 - 8584. [Abstract] [Full Text] [PDF]
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G. Schofield and S. J. Kimber Leukocyte Subpopulations in the Uteri of Leukemia Inhibitory Factor Knockout Mice During Early Pregnancy Biol Reprod, April 1, 2005; 72(4): 872 - 878. [Abstract] [Full Text] [PDF]
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 T. Daikoku, S. Tranguch, D. B. Friedman, S. K. Das, D. F. Smith, and S. K. Dey Proteomic Analysis Identifies Immunophilin FK506 Binding Protein 4 (FKBP52) as a Downstream Target of Hoxa10 in the Periimplantation Mouse Uterus Mol. Endocrinol., March 1, 2005; 19(3): 683 - 697. [Abstract] [Full Text] [PDF]
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J. Li, J. V. Zhang, Y.-J. Cao, J.-X. Zhou, W.-M. Liu, X.-J. Fan, and E.-K. Duan Inhibition of the Beta-Catenin Signaling Pathway in Blastocyst and Uterus During the Window of Implantation in Mice Biol Reprod, March 1, 2005; 72(3): 700 - 706. [Abstract] [Full Text] [PDF]
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K. Y Lee and F. J DeMayo Animal models of implantation Reproduction, December 1, 2004; 128(6): 679 - 695. [Abstract] [Full Text] [PDF]
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X. Hou, Y. Tan, M. Li, S. K. Dey, and S. K. Das Canonical Wnt Signaling Is Critical to Estrogen-Mediated Uterine Growth Mol. Endocrinol., December 1, 2004; 18(12): 3035 - 3049. [Abstract] [Full Text] [PDF]
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