Identification of Indian Hedgehog as a Progesterone-Responsive Gene in the Murine Uterus
Norio Takamoto,
Bihong Zhao,
Sophia Y. Tsai and
Francesco J. DeMayo
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Francesco J. DeMayo, One Baylor Plaza, Houston, Texas 77030. E-mail: fdemayo{at}bcm.tmc.edu.
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ABSTRACT
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Progesterone (P4) plays a central role in normal uterine function, from embryo implantation in endometrium to establishment and maintenance of uterine quiescence during pregnancy in the myometrium. Considering its diverse physiological effects on female reproductive function, rather little is known about downstream events of P4 action. Recent progress in differential screening technologies facilitated identification of such inducible genes. We used uteri of wild-type and progesterone receptor null mutant mice as a starting material and screened for differentially expressed genes by medium-density cDNA expression array. Here, we report that the expression of the morphogen, Indian hedgehog (Ihh), is rapidly stimulated by P4 in the mouse uterus. The level of Ihh mRNA is induced within 3 h, after a single administration of P4 to ovariectomized mice. The induced Ihh mRNA and protein were localized to the luminal and glandular epithelial compartment of the endometrium. During pseudopregnancy, the Ihh mRNA level was transiently increased in the preimplantation period and d 3 and d 4 post coitum and then decreased rapidly at d 5 post coitum. Furthermore, the expression profile of patched-1, hedgehog interacting protein-1, and chicken ovalbumin upstream promoter-transcription factor II, genes known to be in the hedgehog signaling pathway in other tissues, followed the expression pattern of Ihh during the periimplantation period. Our results suggested that Ihh is regulated by P4, and the Ihh signaling axis may play a role in the preparation of the uterus for implantation during the periimplantation period.
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INTRODUCTION
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EMBRYONIC IMPLANTATION IN mammals requires the synchronization of the ability of the uterus to support embryo implantation with the developmental stage of the embryo (1). This synchrony is achieved by endocrine signaling between the ovary and uterus, with the ovarian steroids, 17ß-estradiol (E2) and progesterone (P4), playing a major role in the preparation of the uterus for implantation (2). Many aspects of embryo implantation are species specific. However, of the diverse animal models used to investigate the process of implantation, the mouse has emerged as a model in which the molecular mechanisms regulating this process can be addressed by in vivo genomic manipulation (3). In the mouse, the periimplantation period can be divided into prereceptive, receptive, and refractory with implantation occurring at d 4 of pregnancy. Uterine development of the prereceptive period is under the influence of P4 stimulation. During this P4-dominated period, a nidatory surge of E2 is required to confer endometrial receptivity to the sensitized uterus, facilitating the implantation (4). The action of the steroids P4 and E2 is mediated by the nuclear hormone receptors progesterone receptor (PR) and estrogen receptor-
and -ß, respectively. In the mouse, embryo attachment is followed by a characteristic endometrial transformation, decidualization, which involves both differentiation and cell proliferation. PR action is indispensable (5), whereas estrogen receptor-
is not essential (6) for this process. In the mouse, P4 appears to prime the endometrium before embryo attachment/implantation, and thus, it is one of the essential functions regulated by P4 during the periimplantation period.
P4 action is mediated by the binding of the steroid to its cognate receptor, the PR. The PR is a member of a family of nuclear receptors, which are transcription factors that regulate gene transcription in response to the binding of the receptor to its specific ligand. The mouse PR consists of two isoforms, PR-A and PR-B. PR-B consists of an additional 160 amino acids on the amino terminus of PR-A (7). The two isoforms are a result of differential usage of two in-frame translation initiation sites. Both isoforms of the murine PR gene have been ablated (5). Furthermore, using site-directed mutagenesis in embryonic stem cells, the PR-A isoform has been specifically ablated (8). These mice have been termed PRKO (PR knockout) and PRAKO (PR-A knockout), respectively. The PRKO mouse demonstrated that PR regulates all aspects of female reproduction. In the uterus, ablation of PR rendered the uterus incapable of undergoing a decidual reaction. When the PRKO mice were placed under chronic estrogen stimulation, increased hyperplasia and an inflammatory response were observed (5). The PRAKO female was also incapable of eliciting a decidual response to steroid stimulation, demonstrating that the PR-A isoform was responsible for the regulation of uterine stroma differentiation during implantation. Therefore, these mice serve as useful tools by which to validate the role of PR in the mouse uterus (9).
Although much is known about the molecular mechanisms of PR action and the physiological role of PR in reproduction, little is known regarding the downstream target genes of PR activation. In an attempt to gain more insights about downstream events of P4/PR action in the endometrium, we initiated medium-density cDNA array screening to identify differentially expressed genes in wild-type and PRKO uterus after acute treatment with P4. This approach allowed the screening of 1200 known genes for their ability to be differentially regulated, specifically by the PR. One of the genes identified as being differentially expressed was Ihh.
Ihh is a member of the developmentally regulated morphogens, the Hedgehog (Hh) gene family (10). The Hh family of proteins was first cloned in Drosophila and has been extensively studied in both Drosophila and higher vertebrates. There are three Hh homologs in mice, namely Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) (10). In general, Hh signaling regulates both cell proliferation and differentiation by either short-range or long-range actions, as Hh protein is diffusible. This is involved in signaling between two distinct compartments (e.g. epithelium and mesenchyme) (10). The vertebrate Shh acts on a variety of developmental processes including the development of the ventral neural tube, ventral somites (11), left-right asymmetry (12), foregut development (13), polarizing activity of the limb (14), and lung development (15, 16). Dhh is important for spermatogenesis in the testis (17, 18). Ihh has been reported in the regulation of bone development (19, 20), gastrointestinal tract development (21), and embryonic vasculogenesis (22). Regulation of Hh signaling and its function by steroid hormones in the endometrium have not been established previously. Targeted null mutation of Ihh has been made and exhibited perinatal lethality, presumably caused by respiratory failure due to malformation of the thorax (23). Therefore, the role of Ihh in uterine physiology could not be evaluated. In the present study, we have identified Ihh as a downstream target of P4/PR in the uterus. Ihh is acutely induced in luminal epithelium (LE) and glandular epithelium (GE) by P4. Ihh exhibited a dynamic spatio-temporal expression pattern in the uterus during the periimplantation period. Moreover, we demonstrated that the expression pattern of known genes of the Hh signaling pathway are expressed in a similar pattern to Ihh in the pseudopregnant mouse uterus. Our results indicate that uterine Ihh is acutely up-regulated by P4 and the Hh signaling is operable during the periimplantation period, which may exert its function by P4-initiated epithelial-mesenchymal interaction during the periimplantation period.
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RESULTS
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Ihh Is Identified as Differentially Regulated by P4
Medium-density cDNA array (Atlas cDNA Expression Arrays, CLONTECH Laboratories, Inc., Palo Alto, CA) was used to identify differentially expressed genes between wild-type and PRKO uteri. In an attempt to identify rapidly induced genes by P4, ovariectomized mice (both wild-type and PRKO) were treated with 1 mg of P4 (sc) and killed 6 h later. Total uterine RNA was used to synthesize an array of gene-specific cDNA probes to be hybridized with the mouse 1.2 k-I array (Fig. 1A
). Many genes were shown to be differentially expressed. Among these genes, the expression level of Ihh was 6.1-fold lower in PRKO uterine RNA as compared with wild-type uterine RNA. This suggested that ablation of PR function in the presence of P4 affects Ihh mRNA expression. The result of the DNA microarray was validated by ribonuclease (RNase) protection assay (RPA). RPA was performed on uteri from wild-type and PRKO mice (Fig. 1B
). RPA demonstrated lower levels of Ihh mRNA in PRKO samples as compared with wild-type samples. These results are consistent with the finding of the DNA microarray.

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Figure 1. Identification of Ihh as a Differentially Expressed Gene in PRKO Uterus
A, Ovariectomized mice were treated with vehicle or P4 for 6 h. The atlas cDNA expression array was hybridized with wild-type probes (+/+) or PRKO probes (-/-). Arrow indicates signals for Ihh. B, RPA of Ihh is shown. Cyclophilin (Cph) serves as an internal standard to ascertain equivalent loading. Ovariectomized wild-type and PRKO mice were treated with P4 for 6 h. Total RNA (10 µg) was used per reaction. Results of RPA are representative of three independent experiments.
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The above analysis demonstrated that Ihh is differentially expressed in the uteri of PRKO and wild-type mice after acute treatment with P4. Because PR action has been impaired in PRKO mice, this could be due to the alteration of uterine development that may require PR (24, 25, 26). To demonstrate that differential expression of Ihh is a result of P4 stimulation of gene expression, ovariectomized wild-type female mice (6 wk old) were treated with either vehicle (sesame oil) or P4, (1 mg, sc) and killed 6 h after the commencement of treatment. Total RNA from uteri was assayed by RPA for the expression of Ihh, as well as for the known P4-responsive genes, calcitonin and PR. The level of mRNA of calcitonin is known to be up-regulated (27) and that of PR is down-regulated (28) by P4. As expected, the levels of Ihh mRNA were induced by acute P4 treatment as was the other P4-responsive gene, calcitonin (Fig. 2
). As expected, expression of the PR mRNA was decreased after P4 stimulation.

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Figure 2. P4 Responsiveness of Ihh Gene Expression in the Uterus
RPA for Ihh after 6 h of treatment with P4 in wild-type ovariectomized uterus is shown. Cal, Calcitonin; Cph, cyclophilin. Results of RPA are representative of three independent experiments.
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Localization of Ihh Induction in the Mouse Uterus
In situ hybridization analysis and immunohistochemical analysis was used to identify the compartment in which Ihh was induced in the mouse uterus. Wild-type mice were ovariectomized and treated with P4 for 6 h. In situ hybridization analysis (Fig. 3
, A and B) and immunohistochemical analysis (Fig. 3
, C and D) demonstrated that Ihh mRNA and protein were induced in LE and GE of P4-treated wild-type uterus. Treatment with P4 did not induce Ihh protein in PRKO uteri, and in situ hybridization analysis with sense probe or immunohistochemical analysis with nonimmunized serum substituted for primary antibody showed no signal (not shown). Thus, Ihh mRNA and protein were induced by acute treatment with P4 in the epithelial compartment.

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Figure 3. P4 Induction of Ihh mRNA and Protein in the Endometrial Epithelium
Induction of Ihh mRNA was localized by in situ hybridization. Ovariectomized female mice were treated with vehicle alone (A), or with P4 (1 mg, B) for 6 h. Ihh protein expression was examined by immunohistochemistry. Vehicle alone and P4 (1 mg)-treated samples are shown in panels C and D, respectively. Photomicrographs shown are representative of four independent experiments (Bar, 100 µm).
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Temporal Induction of Ihh by P4 in the Mouse Uterus
To assess the temporal induction of Ihh by P4, ovariectomized wild-type female mice were stimulated with a single dose of P4 (1 mg) and killed at 0, 3, 6, 9, 12, 15, and 24 h post injection. The expression levels of Ihh and two other uterine epithelial P4-responsive genes, amphiregulin (29) and calcitonin (27) were measured by RPA. As shown in Fig. 4
, 3 h after commencement of P4, Ihh mRNA was increased from undetectable levels at 0 h to detectable at 3 h, peaked between 69 h, and decreased thereafter. Both amphiregulin and calcitonin induction by P4 could be detected by 6 h, with peak induction of amphiregulin at 69 h and calcitonin at 1215 h. Lactoferrin, an estrogen-responsive gene (30), did not show any induction over this time course (not shown). This analysis demonstrates that Ihh mRNA was rapidly induced by P4. Examination at earlier time courses shows significant induction of Ihh by 2 h (data not shown). In addition, Ihh induction by P4 appeared to precede induction of previously identified P4-responsive genes, amphiregulin and calcitonin.

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Figure 4. Kinetics of P4 Induction of Expression of Ihh, Amphiregulin, and Calcitonin
Ovariectomized wild-type mice were treated with P4 (1 mg), and uteri were dissected at the time indicated (hours). RPA using total RNA (10 µg per reaction) was conducted. Ihh mRNA levels were induced at approximately 3 h and peaked around 6 h. Amphiregulin (Ar) was induced at about 6 h and peaked around 9 h. Calcitonin (Cal) was induced at 6 h and peaked around 1215 h. Data shown are representative of four independent experiments using two mice at every time point.
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Expression Profile of Ihh mRNA During Pseudopregnancy
The analysis, thus far, demonstrated that Ihh is induced in the epithelial cells in response to acute supraphysiological treatment with exogenous P4 in an ovariectomized mouse model. To assess the physiological regulation of Ihh, the expression profile of Ihh during the periimplantation period was investigated using a pseudopregnancy model. The levels of Ihh mRNA were determined by RPA. At d 1, mRNA of Ihh was undetectable, increased to detectable levels at d 2, and peaked at d 34, and decreased thereafter (Fig. 5
). Ihh expression profiles temporarily proceed to the other known P4-regulated implantation markers, such as amphiregulin and calcitonin.

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Figure 5. Expression Profile of Ihh, Amphiregulin, and Calcitonin during Pseudopregnancy
RPA was performed using total RNA (10 µg) isolated from wild-type pseudopregnant uteri at the days indicated. Pseudopregnancy was begun by mating vasectomized males with superovulated females, and the day of copulation plug was designated as d 1. Total RNA was isolated from d 1d 7 of pseudopregnancy (D1D7). Ar, Amphiregulin; Cal, calcitonin; Cph, cyclophilin. Cph serves as an internal control to ascertain equivalent loading of the RNA. Data represent four independent experiments.
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Having demonstrated by RPA that Ihh expression peaks before the implantation period, in situ hybridization analysis was conducted to further confirm that Ihh expression was induced in the endometrial epithelial compartment. The induced cellular localization of Ihh was also compared with the localization of PR. The PR protein was detected in both nuclei of LE and GE, and was weakly expressed in stromal cells as detected by immunohistochemical analysis at d 2 and d 3 of pseudopregnancy (Fig. 6
, A and B). At d 6, immunoreactive PR was down-regulated in the LE and the GE and was mainly detected in periluminal stroma (Fig. 6C
). Examination of the expression pattern of Ihh during the periimplantation period by in situ hybridization demonstrated that Ihh was induced in the LE and GE, which was visible at d 2 (Fig. 6D
), and strong signals were detected at d 3 (Fig. 6E
). At d 6, Ihh expression decreased significantly (Fig. 6F
). Thus, Ihh is induced before the implantation window in the pseudopregnant model, and the timing of initial induction in the epithelial cells coincides with the expression of PR in the epithelial cells of the endometrium.

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Figure 6. Expression Profile of PR Protein and Ihh mRNA During Pseudopregnancy
Immunohistochemistry of PR (AC) and in situ hybridization of Ihh (DF) are shown. Uteri were collected at d 2 (A and D), d 3 (B and E), and d 6 (C and F) of pseudopregnancy. Representative photomicrographs from four independent animals at each time point are shown (Bar, 100 µm).
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Expression of Members of the Ihh Signaling Pathway During the Preimplantation Period
In vertebrates, the signaling pathway of Hh consists of a variety of components of proteins, such as ligands (Shh, Ihh, Dhh), membrane receptors (patched: Ptc-1, Ptc-2), intracellular signal transducer (smoothened: Smo), Zn finger-containing Gli family of transcription factors (31), and a negative regulator (hedgehog interacting protein: HIP-1) (32). Of these members of the Hh signaling cascade, expression of Ptc-1 and HIP-1 are regulated by Hh (10). Finally, a member of the nuclear orphan receptor family, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), expression in mice is known to be regulated, at least in part, by a member of the Hh family, Shh. A Shh response element, ShhRE has been identified in the COUP-TFII promoter (33, 34). Therefore, we investigated, by in situ hybridization, the pattern of expression of Ptc-1, HIP-1, and COUP-TFII during pseudopregnancy to determine whether these members of the Hh signaling pathway are also differentially regulated during this period.
Ptc-1, a receptor for Hh protein, is induced by Hh signaling. In the uterus, Ptc-1 expression was undetectable at d 2 (Fig. 7A
). At d 4, epithelial cells and periepithelial stroma were stained positively (Fig. 7B
). At d 6, localized Ptc-1 expression was down-regulated (Fig. 7C
), and weak staining was detected in the mesometrial pole of the LE (not shown). Weak HIP-1 staining was shown in the LE and the GE at d 2 (Fig. 7 D
), but increased HIP-1 staining was detected in both the LE and periluminal stroma at d 4 (Fig. 7E
), and at d 6, became undetectable (Fig. 7F
). Similarly, weak staining of COUP-TFII was observed in the LE and periluminal stroma at d 2 (Fig. 8G
). At d 4, COUP-TFII mRNA was up-regulated in stromal cells and epithelial cells (Fig. 8 H
). At d 6, COUP-TFII expression was down-regulated (Fig. 8I
), and localized expression in the mesometrial pole of the LE was observed, which resembles Ptc-1 expression at d 6 (not shown). Thus, Ihh mRNA and its downstream target genes, Ptc-1, HIP-1, and COUP-TFII, are temporarily regulated during the preimplantation period in close association with Ihh expression.

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Figure 7. Expression Profile of Hh Target Genes during Pseudopregnancy
In situ hybridization of Ptc-1 (AC), HIP-1 (DF), and COUP-TFII (GI) are shown. Uteri were collected at d 2 (A, D, and G), d 4 (B, E, and H), and d 6 (C, F, and I) of pseudopregnancy. Representative photomicrographs from four independent animals at each time point are shown (Bar, 100 µm).
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Figure 8. Regulation of Ihh and Downstream Target Genes in Endometrium
A model of PR-Ihh signaling in endometrium is shown. For detail see text.
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DISCUSSION
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Identification of P4-Regulated Genes Using Microarray Strategy and PRKO Mice
The use of the medium-density cDNA microarray technology, in combination with the PRKO mouse, allowed the identification of Ihh as a downstream target of P4 action. There were several advantages of the use of the PRKO mouse in this approach. First, PRKO uteri do not express functional PR, and whereas acute treatment with P4 induces responsive genes in wild-type uterus, in the PRKO uterus, PR-dependent gene expression is unaffected, thus augmenting the differential gene expression between the two genotypes. This optimized differential expression is important for accurate data acquisition and interpretation for array analysis. Second, the power of using the PRKO mouse model allowed exclusion of yet unclear nongenomic effects by P4 that might cause change in gene expression. For example, P4, independent of PR, can affect the calcium-dependent signaling cascade that is evoked by the G protein-coupled receptor (35). Nongenomic effects are excluded from our model, because both wild-type and PRKO uteri were treated in the same way, such that P4-dependent and PR-independent activity, which may rapidly affect gene expression, is subtracted.
The goal of this microarray approach was to detect primary targets of PR activation. To accomplish this goal and avoid the detection of secondary targets of P4 action, an acute treatment of ovariectomized mice with P4 was employed. This study demonstrated a rapid induction of Ihh by P4 similar to, if not sooner than that observed for, amphiregulin and calcitonin (27, 29). This acute induction of Ihh is not due to a nongenomic effect of P4 because it is not induced in PRKO uterus in the presence of P4. Genomic analysis of the sequences flanking the mouse and human Ihh gene identified several putative P4 response elements. However, strict mapping of a transcription start site has yet to be accomplished.
To identify an appropriate cell culture system to investigate the activation of Ihh by P4, two steroid-responsive endometrial carcinoma cell lines, Ishikawa cells (36) and RL952 cells (37), were tested for acute P4 stimulation of Ihh expression. Unfortunately, Northern blot and RT-PCR analysis failed to detect any specific signals (data not shown), indicating that these cell lines fail to induce Ihh expression upon P4 treatment. Thus, these cell lines are not appropriate for studying the regulation of Ihh by P4, and precise analysis of 5'-flanking region of Ihh in response to P4 must await mapping of 5'-flanking region and identification of a cell line that mimics the in vivo response. Nonetheless, although our results do not definitively prove that Ihh is a direct target of PR activation, they strongly indicate that Ihh is a promising candidate for the primary response gene of P4/PR. The direct ligand-dependent regulation of Ihh gene transcription by PR still must be demonstrated by systematical in vitro promoter analysis in an appropriate cell line.
Expression of the Ihh Signaling Axis During the Preimplantation Period
We investigated three known downstream target genes of Hh signaling, Ptc-1, HIP-1, and COUP-TFII, during periimplantation. Ptc-1 and HIP-1 belong to the Hh signaling pathway and are induced by Hh (10). Ptc-1 is a receptor for Hh. Therefore, we thought it was necessary to confirm its localization and expression pattern to ascertain whether uterine Ihh is functional. Increased Ihh mRNA at d 3 was followed by Ptc-1 expression in the stromal cells, thus implicating epithelial-mesenchymal interaction. HIP-1 showed a similar pattern of induction. Because HIP-1 negatively regulates Hh signaling (32), its concomitant induction during the periimplantation period might function to ensure precise control of Hh signaling in preimplantation endometrium. Because Ihh is the only member of the Hh family expressed in the mouse uterus (38), this coordinated expression of members of the Hh pathway would indicate regulation of these members by Ihh. Our results of Ihh expression in a pseudopregnant model are consistent with a description in a previous report using timed pregnant mouse uterus (38).
COUP-TFII is a nuclear orphan receptor that belongs to a nuclear hormone receptor superfamily (39). COUP-TFII is highly expressed during embryonic development, and a null mutant exhibited embryonic lethality (
E10) due to defective angiogenesis and inflow tract development (40). There are two highly homologous COUP-TFs, COUP-TFI and II, in mouse and they may play a redundant role (39). In the uterus, COUP-TFII is highly expressed whereas COUP-TFI is undetectable by RPA using specific riboprobes (data not shown). We chose COUP-TFII as an endometrial stromal cell marker for Ihh signaling for the following reasons. First, COUP-TFII is a downstream target gene of Hh signaling in vivo and in vitro. COUP-TFII is highly expressed in motor neurons during development, and ectopic expression of COUP-TFII was induced by transplantation of notochord which secretes Shh (41). The Shh-responsive element within the 5'-flanking region of COUP-TFII had been identified using P19 mouse embryonic carcinoma cells. This element is distinct from the Gli response element and requires protein phosphatase activity (33, 34). Next, the COUP-TFII haploinsufficiency phenotype, caused by a deletion of a single allele of COUP-TFII in mice, demonstrated reduced fecundity in females (40). This phenomena is in part associated with altered uterine functions. COUP-TFII expression and function in the normal endometrium and abnormal endometrium (endometriosis and endometrial cancer) have been reported previously (42, 43, 44, 45, 46). With this in mind, we hypothesized that one of the pathways potentially regulated by Ihh (via PR) might involve COUP-TFII. Concomitant temporal up-regulation of COUP-TFII is consistent with this hypothesis. COUP-TFII is expressed in a variety of adult organs, no cognate ligand has been identified, and null mice are embryonic lethal. Therefore, specific analysis of the uterine functions of COUP-TFII should wait for the development of uterus-specific conditional COUP-TFII mutants.
Potential Role of Ihh Signaling During Periimplantation
A growing number of studies regarding Hh signaling using genetically modified mice indicate that these molecules play essential roles in a variety of physiological processes, not only during development (19, 23, 47, 48, 49). This evidence might allow us to speculate on a potential role of Ihh in the uterus. Physiological functions of Ihh are well studied in bone. In the regulation of bone formation, Ihh signaling promotes cell proliferation and cell differentiation, in part, by coordinating the expression of PTHrP (23, 47, 48), bone morphogenic proteins, BMP2 and 4, noggin, and chordin (19). In the female steroid hormone-responsive tissue, Hh signaling is known to play a role in the mammary gland. Ihh is expressed in mammary gland, and experimental enhancement of Hh signaling in the mammary gland by conditional haploinsufficiency of Ptc-1 resulted in mammary ductal hyperplasia (49). Disruption of the Ihh target, PTHrP, also resulted in disruption of mammary gland development (50, 51). The downstream target genes of Ihh in the bone, such as PTHrP, BMP-2, -4, -5, and -7, and noggin, are all expressed in the uterus (38). Thus, it might be reasonable to speculate that the Ihh signaling axis in the bone and mammary gland is conserved in the uterus and regulates endometrial epithelial and stromal cell proliferation and differentiation. Ihh may also play a role in uterine vascularization during the preimplantation period. It has been demonstrated that Ihh is a primitive endoderm-secreted signal that alone is sufficient to induce formation of hematopoietic and endothelial cells (22). Importantly, ablation of COUP-TFII resulted in early embryonic lethality due, in part, to disruption of angiogenesis (40). Recently, it has been demonstrated also that P4 stimulates angiogenesis in the uterus (52). Although it still remains speculative, one potential role of Ihh is to mediate P4 stimulation of angiogenesis in the uterus through the regulation of the expression of COUP-TFII.
COUP-TFII has been implicated to play a role in mesenchymal-epithelial interaction during organogenesis and angiogenesis (39). Regulation of mesenchymal gene expression such as COUP-TFII by P4-regulated epithelial factor, Ihh, might point to a novel paradigm for the steroid regulation of endometrial function. This epithelial-mesenchymal interaction, acutely initiated by P4 before implantation, had been envisioned. Amphiregulin, which is an epidermal growth factor-like protein, is rapidly induced by P4 in the endometrial epithelial cells, in close association with cessation of epithelial cell proliferation (autocrine) and onset of stromal cell proliferation (paracrine) (29). Consistent with this, PR was localized in the epithelium before the implantation window. Therefore epithelial PR initiates epithelial-mesenchymal interaction, before decidual reaction that is initiated by blastocyst attachment in normal pregnancy (38). Thereafter, PR expression shifts to stromal cells, which undergo decidual transformation (53). Decidualization of the endometrium has been divided into three phases (54). In the first phase, uterine stromal cells are sensitized to a decidualizing stimulus. This sensitization requires P4 treatment (54). The second phase is characterized by induction of stromal cell differentiation. Finally, the third phase consists of maintenance of the growth and development of decidualized cells. Our hypothesis is that P4 induction of epithelium-secreted morphogen regulate both proliferation and differentiation in the mesenchyme. We propose that Ihh is one of the factors induced during the first phase of decidualization, which may act to prime the endometrium for decidualization by regulating the downstream target genes that are required for latter phases of decidualization. Consequently, identification and characterization of the downstream target of Ihh signaling in the uterus are important to validate this hypothesis.
Based on all of the above results, we propose the following working model (Fig. 8
). In this model, P4 induces Ihh in the epithelial cells of endometrium and Ihh signaling into the stromal cells. Induction of the target genes in epithelial cells may also have significant autonomous roles. Ihh-mediated epithelial-mesenchymal interaction might induce downstream genes including COUP-TFII, as well as genes yet to be identified. Such downstream gene products may play a role during the periimplantation period, which involves both cell proliferation, differentiation, and angiogenesis (e.g. decidualization).
Further studies are required to substantiate the physiological significance of Hh signaling. A targeted null mutation of Ihh has been made. However, the Ihh null mutants exhibited short-limbed dwarfism and perinatal lethality, which was presumably caused by respiratory failure due to malformation of the thorax (23). Thus the function of Ihh in other organs, such as uterus, has yet to be defined. Future development of in vivo model systems, such as transgenic mice, to overexpress HIP-1 in epithelial cells to attenuate Hh signaling or conditional knockout of Ihh in the uterus, might be necessary for assessing the role of Ihh signaling in the prereceptive endometrium.
Thus, Ihh is an important factor that regulates a variety of developmental and physiological processes (e.g. in bone, mammary gland, and intestine), and its regulation by P4 in the endometrial epithelial cells might be shedding new light on the P4-regulated endometrial functions through epithelial-mesenchymal interaction before implantation.
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MATERIALS AND METHODS
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Animals
The PRKO mice were generated and described previously (5). Mice were maintained in the designated animal care facility at the Baylor College of Medicine according to the institutional guideline for the care and use of laboratory animals. For P4 treatment, 2 wk before the commencement of the treatment, bilateral ovariectomy was conducted and P4 (1 mg/0.1 ml sesame oil per animal) was injected sc at 6 wk of age. Six hours after P4 injection (or indicated time for time course study), animals were anesthetized with Avertin (2,2,2-tribromoethyl alcohol, Sigma-Aldrich Corp., St. Louis, MO) and killed by cervical dislocation. Wild-type females for pseudopregnancy were mated with vasectomized males after superovulation (pregnant mares serum gonadotropin, 5 IU, and human chorionic gonadotropin, 5 IU, Sigma), and the morning of vaginal plug was designated as d 1.
RNA Preparation and RPA
Total RNA was isolated using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). Briefly, after the dissection, uteri were cleared from adipose tissues, and were immediately frozen on dry ice and stored at -80 C. Frozen samples were homogenized in TRIzol, and the total RNA was isolated according to the protocol provided by the company.
RPA was performed as described previously (55) using a commercially available kit (RPAII, Ambion, Inc., Austin, TX).
Screening of Differentially Expressed Genes
Total RNA from both wild-type and PRKO uteri were treated with RNase free-deoxyribonuclease to minimize genomic DNA contamination. Subsequently, mRNA was enriched using biotinylated-oligo(dT) and streptoavidin-magnet beads from CLONTECH Laboratories, Inc. Enriched poly A-tailed RNA was isotopically labeled (
-32P dATP) by reverse transcription reaction and separated from unincorporated nucleotides using a gel filtration column. The expression array membrane was hybridized with probes for 16 h at 68 C. After a high-stringency wash of the hybridized array, blots were subjected to quantitative analysis by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The specific signals were quantified and fold induction was calculated after normalization using Atlas Vision 3.0 software (CLONTECH Laboratories, Inc.).
Riboprobes
For RPA, 32P-UTP-labeled antisense RNA was synthesized using a commercial in vitro transcription kit (Ambion, Inc.) with linearized template and appropriate RNA polymerase.
For nonisotopic in situ hybridization, digoxigenin-labeled riboprobes were generated using DIG-RNA labeling kit (Roche Diagnostics, Indianapolis, IN).
The cDNA probes for Ihh, HIP-1, were generated by RT-PCR and subcloned into a pCRII-TOPO cloning vector using a kit (Invitrogen, Carlsbad, CA). The sequence of oligonucleotides used to generate cDNA probes are as follows: Ihh, for RPA: forward 5'-CTG TGA ACT GAG CTG ACA AGC GTG-3', reverse 5'-GGG AAT CTA GCA GCA TCG ACT GAG-3', and for in situ hybridization: forward 5'-CCG CGT TGC CAA AAC AAA CG-3', reverse 5'-CCA GGA AAA TAA GCA CAT CA-3'. This probe generated exactly the same result as the probe obtained from Dr. Andrew McMahon (Harvard University, Cambridge, MA). HIP-1, for RPA: forward 5'-GTG GCT CTG GGC TTC TTT GAA GG-3', reverse 5'-GCA CAT TGG ATC TCC TCC AGC AGC-3'. For in situ hybridization forward 5'-CGC CCT TTC GGT TCC TGC TAC TGT C-3', reverse 5'-GCA CAT TGG ATC TCC TCC AGC AGC-3'. Probes for calcitonin and amphiregulin were previously described. Probes for PR was obtained from Dr. Jeff Rosen (Baylor College of Medicine, Houston, TX). Probes for Ptc-1 were from Dr. Matthew P. Scott (Stanford University, Stanford, CA). The cDNA template for cyclophilin was purchased from Ambion, Inc. A probe for COUP-TFII was described previously (40).
Tissue Preparation for Histology
The small portions (
5 mm in length) of uteri were fixed in freshly prepared 4% paraformaldehyde in diethyl pyrocarbonate-treated PBS immediately after the dissection for histological evaluation. After overnight fixation at room temperature, tissues were dehydrated through series of ethanol and then processed for paraffin embedding using an automated tissue processor (Shandon Lipshaw, Pittsburgh, PA). Paraffin sections (8 µm) were mounted onto poly-L-lysine-coated slides (VWR Scientific Products, West Chester, PA), and then used for subsequent applications.
Immunohistochemistry
Immunohistochemistry for Ihh was performed by indirect immunoperoxidase staining. After dehydration through graded alcohol, tissue sections were quenched by 3% hydrogen peroxide in absolute methanol for 15 min and then treated with 1x trypsin (Sigma) for 15 min at room temperature, followed by blocking with 10% normal rabbit serum in PBS. The primary antibody against Ihh (goat polyclonal, Research Diagnostics, Flanders, NJ) at 1:200 were incubated on the pretreated sections for 1 h at room temperature in a humidified chamber. Sections were then treated with biotinylated rabbit antigoat IgG (Accurate Chemicals and Scientific Corp., Westbury, NY), and streptoavidin-horseradish peroxidase conjugate (Zymed Laboratories, Inc., South San Francisco, CA) both at 1:1000 dilution, followed by color development using 3,3'-diaminobenzidine as a chromogen. Sections were counterstained with methyl green (Sigma) and mounted with Permount (Fisher Scientific, Pittsburgh, PA).
PR Immunohistochemistory was performed according to the protocol (8) of Dr. John P. Lydon (Baylor College of Medicine).
In Situ Hybridization
Nonisotopic in situ hybridization was conducted by the published protocol from Dr. David Andersons laboratory (http://www.muridae.com/wmc/docs/Big_In_Situ.html) with a modification for paraffin sections. Briefly, paraffin sections were dehydrated and fixed with 4% paraformaldehyde. Pretreatment included incubation with 10 mM HCl, acetylation with 0.5% acetic anhydride, and proteinase K treatment. Hybridization (16 h) and high-stringency wash (30 min, twice) was performed at 65 C in the presence of 50% formamide, followed by an extensive wash with 1x SSC (0.15 M sodium chloride; 0.015 M sodium citrate, pH 7.0) at room temperature. After the hybridization, sections were incubated with antidigoxigenin alkaline phosphatase conjugate (1:1000, Roche Diagnostics), and chromogenic detection with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Life Technologies, Inc.) was performed in buffer consisting of 100 mM Tris (pH 9.5), 50 mM MgCl2, 100 mM NaCl. Negative staining was confirmed for all the probes used in this study by sense probe and/or RNaseA-treated sections.
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ACKNOWLEDGMENTS
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We thank Meirong Gu, Sandy Ma, Janet L. DeMayo, and Xiaoyan Huang for excellent technical assistance. We thank Drs. Andrew McMahon, Matthew P. Scott, and Jeff Rosen for providing cDNA probes; Dr. John P Lydon for antibody and protocol for PR immunohistochemistry; Drs. Carolyn Smith, Ann Word, and Jesse Smith for endometrial carcinoma cells; and John Ellsworth for preparation of this manuscript.
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FOOTNOTES
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This work was supported by Wyeth-Ayerst Laboratories, Inc., an NIH Grant (HD-42311) to F. J. D., and NIH grants to S.Y.T. (DK-55636 and DK-57743, project 4).
Abbreviations: COUP-TFII, Chicken ovalbumin upsteam promoter-transcription factor II; Dhh, Desert hedgehog; E2, 17ß-estradiol; GE, glandular epithelium; Hh, hedgehog; HIP-1, hedgehog interacting protein 1; Ihh, Indian hedgehog; LE, luminal epithelium; P4, progesterone; PR, progesterone receptor; PRKO, PR knockout; Ptc-1 and -2, patched 1 and 2; RNase, ribonuclease; RPA, ribonuclease protection assay; Shh, Sonic hedgehog.
Received for publication July 20, 2001.
Accepted for publication June 14, 2002.
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