help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0283
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hayashi, K.
Right arrow Articles by Spencer, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hayashi, K.
Right arrow Articles by Spencer, T. E.
Endocrinology Vol. 148, No. 7 3496-3506
Copyright © 2007 by The Endocrine Society

WNTs in the Ovine Uterus: Potential Regulation of Periimplantation Ovine Conceptus Development

K. Hayashi, R. C. Burghardt, F. W. Bazer and T. E. Spencer

Center for Animal Biotechnology and Genomics (K.H., R.C.B., F.W.B., T.E.S.), Department of Animal Science (K.H., F.W.B., T.E.S.), and Image Analysis Laboratory and Department of Veterinary Integrative Biosciences (R.C.B.), Texas A & M University, College Station, Texas 77843

Address all correspondence and requests for reprints to: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A & M University, College Station, Texas 77843-2471. E-mail: tspencer{at}tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
WNTs (Wingless-type MMTV integration site family member) are involved in critical developmental and growth processes in animals. These studies investigated WNT pathways in the ovine uterus and conceptus during the periimplantation period of pregnancy. WNT2 and WNT2B mRNAs were detected in endometrial stroma. WNT5A and WNT5B mRNAs were most abundant in the stroma and less so in the luminal epithelium, whereas WNT11 mRNA was detected primarily in the glands. WNT7A mRNA was present in the luminal epithelium on d 10, absent on d 12 and 14, and increased between d 16 and 20. Only WNT2, WNT2B, and WNT4 were detected in conceptus trophectoderm. FZD6/8 (frizzled receptor) and GSK3B (glycogen synthase kinase 3ß) mRNAs were detected primarily in endometrial epithelia and conceptus trophectoderm, whereas the LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6) coreceptor was present in all endometrial cells and the trophectoderm. DKK1 (Dickkopf), a WNT signaling inhibitor, increased in the endometrium from d 16–20. CTNNB1 [catenin (cadherin associated protein) ß1] and CDH1 (E-cadherin) mRNAs were most abundant in the endometrial epithelia and trophectoderm. LEF1 (lymphoid enhancer-binding factor 1) mRNA was expressed primarily in uterine epithelia, whereas TCF7L2 [(transcription factor 7-like 2 (T-cell specific, HMG-box)] was primarily in the conceptus. CTNNB1 and TCF7L2 proteins were both abundant in the nuclei of trophoblast giant binucleate cells. WNT7A stimulated a TCF/LEF-luciferase reporter activity in ovine trophectoderm cells that was inhibited by dominant-negative TCF and Sfrp2 (secreted FZD-related protein 2). WNT7A increased trophectoderm cell proliferation as well as MSX2 (msh homeobox 2) and MYC (myelocytomatosis oncogene) mRNA levels. Wnt5a increased trophectoderm cell migration in a Rho kinase-dependent manner. These results support the hypotheses that canonical and noncanonical WNT signaling pathways are conserved regulators of conceptus-endometrial interactions in mammals and regulate periimplantation ovine conceptus development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WNT (WINGLESS-TYPE MMTV integration site family member) genes are homologous to the Drosophila segment polarity gene wingless (wg). In humans and mice, the WNT family of genes 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 (1). Autocrine or paracrine signaling by WNTs involves a family of 10 frizzled receptors (FZD) (2), which are seven transmembrane G protein-coupled receptors that possess an extracellular cysteine-rich domain for WNT binding (3, 4). LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6) are coreceptors with FZD (5). Secreted FZD-related proteins (SFRPs) are forms of FZDs that contain the cysteine-rich domain but no transmembrane or intracellular segments and can bind WNTs to inhibit their activity (6, 7). Other antagonists of the WNT signaling pathway are products of four DKK (Dickkopf) genes that encode secreted proteins that bind the LRP coreceptors (8).

Signaling pathways activated by WNTs are generally divided into the canonical and noncanonical or planar cell polarity pathways (9). In the canonical WNT signaling pathway, FZD receptors transduce a signal to several intracellular proteins that include DVL (disheveled), GSK3B (glycogen synthase kinase 3 ß), axin, adenomatous polyposis coli, and the transcriptional regulator CTNNB1 [or catenin (cadherin associated protein) ß1] (9, 10). Cytoplasmic CTNNB1 levels are normally low because of continuous proteasome-mediated degradation controlled by GSK3B. When cells receive WNT signals, the degradation pathway is inhibited, resulting in accumulation of CTNNB1 in the cytoplasm and nucleus. In the cytoplasm, CTNNB1 interacts with CDH1 (E-cadherin). Adherens junctions formed by classical cadherin/catenin complexes mediate epithelial organization and function. Nuclear CTNNB1 interacts with transcription factors, most notably members of the TCF7/LEF1 [transcription factor 7 (T-cell specific, HMG-box)/lymphoid enhancer-binding factor one] family, to regulate transcription of genes involved in cell proliferation and differentiation, such as MYC (myelocytomatosis oncogene) and MSX2 (msh homeobox 2). Other WNTs, such as WNT5A and WNT11, appear to primarily activate the noncanonical or planar cell polarity pathway involved in cell movement, proliferation, and migration (9) and may also antagonize the canonical WNT pathways in Xenopus embryos and mammalian cells (11, 12, 13).

WNTs are important regulators of uterine morphogenesis, uterine receptivity to the embryo, and blastocyst implantation in mice (14, 15, 16). Many WNT genes are expressed in human endometrium during the proliferative and secretory phases of the menstrual cycle (17), and the canonical WNT signaling pathway is implicated in trophoblast differentiation in humans (18) and cattle (19). Of particular note, we found that WNT7A is a novel gene induced in the endometrial luminal epithelium (LE) and superficial ductal glands of the ovine uterus by interferon {tau} (IFNT) (19), the pregnancy recognition signal produced by the mononuclear trophectoderm cells of the conceptus (embryo/fetus and associated extraembryonic membranes) from d 11 to 20 (20). No other information is available on WNT signaling pathways in the ruminant uterus and conceptus.

Early pregnancy in sheep is characterized by blastocyst elongation that is concomitant with pregnancy recognition signaling and the onset of trophoblast growth and differentiation (21, 22). The blastocyst hatches from the zona pellucida on d 8, develops into a tubular form by d 11, and then elongates on d 12 to 10 cm or more in length by d 14, and achieves a length of 25 cm or more by d 17. Elongation of the blastocyst is critical for developmentally regulated production of IFNT and conceptus implantation (21). In the sheep conceptus, trophoblast giant binucleate cells (BNCs) begin to differentiate on d 14 (23) and are thought to arise from mononuclear trophectoderm cells by consecutive nuclear divisions without cytokinesis or endoreduplication (24). The BNCs have at least two critical functions, including fusion with uterine epithelial cells to form a hybrid fetomaternal syncytium for successful implantation and subsequent cotyledonary growth in the placentome, and synthesis and secretion of protein, e.g. placental lactogen, and steroid, e.g. progesterone, hormones (24). WNT signaling via CTNNB1 has recently been implicated in bovine trophoblast giant BNC differentiation (19).

Our working hypothesis is that the WNT signaling pathway is a conserved and critical regulator of conceptus-endometrial interactions during establishment of pregnancy in sheep and that canonical and noncanonical WNT signaling is important for trophectoderm growth, differentiation, and function. As a first step in testing our hypothesis, we conducted studies to determine the following: 1) which WNTs and WNT signaling pathways were present in the ovine uterus and conceptus during the periimplantation period of pregnancy; 2) effects of WNT7A on trophectoderm proliferation and gene expression; and 3) effects of WNT5A on trophectoderm migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Crossbred Suffolk ewes (Ovis aries) were observed daily for estrus in the presence of vasectomized rams and were used in the experiments after they had exhibited at least two estrous cycles of normal duration (16–18 d). All experiments and surgical procedures were in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching and approved by the Institutional Animal Care and Use Committee of Texas A & M University. Ewes were maintained according to normal animal husbandry practices.

Experimental design
At estrus (d 0), ewes were mated to an intact ram and then hysterectomized (n = 5 ewes/d) on d 10, 12, 14, 16, 18, or 20 of pregnancy. On d 10 to 16, the uterine lumen was flushed with 20 ml of sterile 10 mM Tris-HCl (pH 8.0) to confirm the presence of one or more morphologically normal conceptuses. Uterine flushes were not obtained on either d 18 or 20 because the conceptus was firmly adhered to the endometrial LE and basal lamina. At hysterectomy, several sections (~0.5 cm) from the midportion of each uterine horn ipsilateral to the corpus luteum were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). In monovulatory pregnant ewes, uterine tissue samples were marked as either contralateral or ipsilateral to the ovary bearing the corpus luteum. No tissues from the contralateral uterine horn were used in this study. After 24 h, fixed tissues were changed to 70% ethanol and then embedded in Paraplast Plus (Oxford Labware, St. Louis, MO). The remainder of the endometrium was physically dissected from myometrium, frozen in liquid nitrogen, and stored at –80 C for RNA extraction.

RT-PCR analysis
Partial cDNAs for components of the WNT signaling pathway were generated by RT-PCR using methods described previously (25) and total RNA isolated from d 16 pregnant endometrium and/or d 18 conceptuses. Primers for each target gene were derived from conserved sequences of human and bovine genes using primer 3 (26). Primer and annealing temperatures used for PCR are summarized in Table 1Go. The amplified PCR products were subcloned into the pCRII cloning vector using a T/A Cloning kit (Invitrogen, Carlsbad, CA) and sequenced in both directions using an ABI PRISM Dye Terminator Cycle Sequencing kit and ABI PRISM automated DNA sequencer (Applied Biosystems, Foster City, CA) to confirm identity.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Sequences of primers for RT-PCR

 
In situ localization of mRNA
In situ hybridization analyses was performed in cross-sections (5 µm) of paraffin-embedded uterine sections (n = 5 ewes/d of pregnancy) using methods described previously (27). Briefly, deparaffinized, rehydrated, and deproteinated cross-sections (5 µm) of the uterine horns from each ewe were hybridized with radiolabeled sense or antisense cRNA probes generated from linearized plasmid DNA templates using in vitro transcription with [{alpha}-35S] UTP. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Eastman Kodak, Rochester, NY), stored at 4 C for 4 d to 1 month, and developed in Kodak D-19 developer. Slides were then counterstained with Gill’s modified hematoxylin, dehydrated through a graded series of alcohol to xylenes, and protected with a coverslip.

Immunohistochemistry
Immunolocalization of CTNNB1 and TCF7L2 [(transcription factor 7-like 2 (T-cell specific, HMG-box)] proteins was performed in cross-sections (5 µm) of paraffin-embedded uterine sections (n = 5 ewes/d of pregnancy) using specific antibodies and a Vectastain ABC Mouse IgG kit (PK-6102; Vector Laboratories, Burlingame, CA). Mouse monoclonal antibodies to mouse CTNNB1 (610153; BD Biosciences, San Jose, CA) and human TCF7L2 antibody (05-511; Upstate Biotechnology, Lake Placid, NY) were used for immunohistochemistry. The working antibody concentrations for immunohistochemistry were 1.0 µg/ml for CTNNB1 and 0.5 µg/ml for TCF7L2. Negative controls were performed in which the primary antibody was substituted with the same concentration of normal mouse IgG (Sigma, St. Louis, MO). Antigen retrieval using a boiling citrate buffer was performed as described previously (28). Multiple tissue sections from each ewe were processed as sets within an experiment. Sections were not counterstained before affixing the coverslip.

Photomicroscopy
Images of representative fields of sections hybridized with antisense or sense cRNAs were recorded under bright-field or dark-field illumination using a Nikon (Tokyo, Japan) Eclipse 1000 photomicroscope fitted with a Nikon DXM1200 digital camera. Representative photomicrographs of protein immunolocalization were also recorded. Constant image acquisition parameters were used across slides to ensure accurate comparisons. Digital photomicrographs were assembled and labeled using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA).

Cell culture
Ovine trophectoderm cells (oTr) were isolated and cultured from d 15 ovine conceptuses and cultured as described previously (29, 30). Briefly, the embryonic disc was removed from the conceptuses, and the remaining tissues were carefully minced, pooled, and placed in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine (BioSource, Camarillo, CA), 700 nM insulin (Invitrogen), 1 mM pyruvate (BioSource), 0.1 mM nonessential amino acids (BioSource), and antibiotics (100 U penicillin, 100 µg streptomycin, and 0.25 µg/ml amphotericin B; Invitrogen). Cultures were maintained in an atmosphere of 5% CO2 in air at 37 C. Fluid-filled trophoblastic vesicles, which developed spontaneously in culture, were physically ruptured to enhance generation of monolayers. One isolate of cells (oTr-1) was used for these studies and displayed typical epithelial cell morphology with both mononuclear cells and a small population of BNCs for more than 20 passages.

Plasmids
The reporter constructs, Super8XTOPFlash that contained eight copies of TCF/LEF binding sites and Super8XFOPFlash that contained eight mutant LEF/TCF binding sites, were generously provided by Dr. Randall Moon (University of Washington, Seattle, WA) (31). Expression constructs for a constitutively active form of human CTNNB1 (codon 33 substitution of tyrosine for serine, S33Y) (32) and a dominant-negative human TCF7L2 (dnTCF) (33) were generously provided by Dr. Bert Vogelstein (Johns Hopkins University Medical School, Baltimore, MD). A full-length human WNT7A cDNA was purchased from Invitrogen. A mouse Sfrp2 cDNA was generously provided by Dr. Sanjoy Das (Vanderbilt University Medical Center, Nashville, TN) (34). Both human WNT7A and mouse Sfrp2 cDNAs were subcloned into the pcDNA3.1 mammalian expression vector from Invitrogen.

Transient transfections and luciferase assays
The oTr-1 cells were subcultured into 12-well plates and maintained in growth medium. At 70% confluency, cells were transiently transfected using ExGen 500 (Fermentas, Hanover, MD) following the instructions of the manufacturer. Luciferase reporter constructs (1 µg/well) were cotransfected with either pcDNA3.1 vector alone or expression plasmids for WNT7A, S33Y CTNNB1, dnTCF, or Sfrp2 (1 µg total per well). After transfection, cells were maintained in growth medium. Some cells were treated with LiCl (20 mM; Sigma), an inhibitor of GSK3B, at 12 h after transfection. Cells were harvested at 24 h after transfection, and luciferase activity was measured using a Promega (Madison, WI) luciferase assay system. Each experiment was independently repeated three times with different batches of oTr-1 cells between passages 7 and 10.

Cell proliferation assay
The oTr-1 cells were subcultured into 12-well plates (50% confluent) in growth medium for 6–8 h and then switched to serum and insulin-free DMEM for 24 h. The cells were then transfected with either pcDNA3.1 vector alone or expression plasmids for WNT7A, Sfrp2, or their combination (1 µg each per well in quadruplicate) using the Exgen 500 (Fermentas) transfection reagent according to the instructions of the manufacturer. The transfected cells were maintained in serum and insulin-free DMEM for 48 h. Cell number was determined as described previously (35). Briefly, DMEM was removed from cells by vacuum aspiration, and cells were fixed in 50% ethanol for 30 min, followed by vacuum aspiration of the fixative. Fixed cells were stained with a Janus Green B in PBS (0.2% w/v) for 3 min at room temperature. The stain was immediately removed using a vacuum aspirator, and the whole plate was sequentially dipped into water and destained by gentle shaking. The remaining water was removed by shaking, stained cells were immediately lysed in 0.5 N HCl, and absorbance readings were taken at 595 nm using a microplate reader. As described previously (35), cell numbers were calculated from absorbance readings using the following formula: cell number = (absorbance – 0.00462)/0.00006926. The entire experiment was independently repeated three times with different batches of oTr-1 cells between passages 7 and 10. Cell number data are expressed as a percentage of cells transfected with the empty pcDNA3.1 vector as a control.

Real-time PCR analysis
The oTr-1 cells were cultured and transfected as described above for the cell proliferation assay. At 48 h after transfection, total cellular RNA (n = 3 wells per construct) was isolated from transfected oTr using the Trizol reagent (Invitrogen) according to the recommendations of the manufacturer. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively. The cDNA was synthesized from total RNA (5 µg) using random primers (Invitrogen), oligo-dT primers, and SuperScript II Reverse Transcriptase (Invitrogen) as described previously (36). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl of water, and stored at –20 C. PCR analysis of mRNA expression was performed using an ABI PRISM 7900HT Fast Real-Time PCR (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems) as the detector according to recommendations of the manufacturer. Primers were designed to amplify cDNAs of less than 100 bp to maximize efficiency and are summarized in Table 2Go. PCR cycle parameters were 95 C for 15 sec and 60 C for 1 min for 40 cycles. Data were analyzed using GeneAmp 7900 SDS software (version 2.2.2). 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. PCR without template or template substituted with total RNA were used as negative controls to verify experimental results. The results are shown as 40 – CT values.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Sequences of primers for real-time PCR

 
Cell migration assay
The oTr-1 cells (50,000 cells/100 µl serum and insulin-free DMEM) were seeded on 8-µm pore transwell inserts (catalog no. 3422; Corning Costar, Corning, NY). Treatments were then added to each well (n = 3 wells per treatment) including the following: serum and insulin-free DMEM (600 µl) as a negative control; recombinant mouse Wnt5a (catalog no. 645-WN; R & D Systems, Minneapolis, MN) at 100, 500, and 1000 ng/ml; 500 ng/ml recombinant mouse Wnt5a with 10, 50, or 100 µM Y27632 [Rho kinase (ROCK) inhibitor; catalog no. 688001; Calbiochem, San Diego, CA]; 500 ng/ml recombinant mouse Wnt5a with 10, 50, or 100 µM JNK (c-Jun N-terminal protein kinase) inhibitor (catalog no. 159-600-R100; Alexis, San Diego, CA); or growth medium including serum and insulin as a positive control. After 8 h, cells on the upper side of the inserts were removed with a cotton swab. For evaluation of migrated cells on the lower surface, inserts were fixed in 4% paraformaldehyde for 3 min. Then, transwell membranes were removed on the slide and counterstained with 4',6'-diamidino-2-phenylindole. The migrated cells were systematically counted in five nonoverlapping locations, which covered approximately 70% of the insert membrane growth area, using a Zeiss (Thornwood, NY) Axioplan 2 fluorescence microscope with an Axiocam HR digital camera and Axiovision 4.3 software. The entire experiment was independently repeated three times with different batches of oTr-1 cells between passages 7 and 10.

Statistical analyses
All quantitative 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 ACTB 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 ≤ 0.05 was considered significant. Data are presented as least-square means with SEs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and analysis of WNT signaling pathways
RT-PCR analyses detected expression of only 7 of the 19 WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT7A, and WNT11) and only 2 of the 10 FZD genes (FZD6 and FZD8) in endometria and/or conceptuses. Expression of LRP5, LRP6, CDH1, GSK3B, CTNNB1, LEF1, TCF4, MSX2, and MYC genes were detected in endometria and/or conceptuses. Only one of the four DKK genes (DKK1) was detected in sheep endometrium by RT-PCR, and none of the six SFRP genes were detected in either endometria or conceptuses.

In situ hybridization and immunohistochemical analyses revealed which cell types in the uterus and conceptus expressed specific WNT signaling pathway components (Figs. 1–3GoGoGo). As illustrated in Fig. 1Go, WNT2 mRNA was detected primarily in the endometrial stroma and at lower abundance in conceptus trophectoderm and increased after d 16. WNT2B mRNA was observed at low abundance primarily in trophectoderm. WNT4 mRNA was particularly abundant in conceptus trophectoderm and at lower abundance in the endometrial stroma, which increased between d 16 and 20 of pregnancy. Between d 10 and 20 of pregnancy, WNT5A mRNA was most abundant in the stratum compactum stroma underlying the LE but was also detected in endometrial epithelia at lower abundance. A similar pattern of expression was found for WNT5B. Neither WNT5A nor WNT5B mRNAs were detected in conceptuses. As expected, WNT7A mRNA was detected only in LE and superficial glandular epithelium (sGE) of the endometrium but was not detected in conceptuses. Distinct temporal alterations in expression of WNT7A mRNA in endometrial LE and sGE were observed. Although WNT7A mRNA was detected in the endometrial LE/sGE of d 10 pregnant ewes, it was absent on d 12 and 14, reappeared on d 16, and then increased to maximal abundance in LE/sGE on d 20. In contrast, WNT11 mRNA was detected predominantly in the endometrial glands and at lower abundance in the LE but not in trophectoderm. Overall abundance of WNT11 mRNA in the endometrium did not change during early pregnancy.


Figure 1
View larger version (95K):
[in this window]
[in a new window]

  		FIG. 1. In situ hybridization analysis of WNTs and FZD receptors in endometria and conceptuses from d 10 to 20 of pregnancy (P). In each column, representative photomicrographs of in situ hybridization results are presented in dark-field illumination. Mel, Melanocyte; S, stroma; Tr, trophectoderm. Scale bar, 100 µm.

 

Figure 2
View larger version (106K):
[in this window]
[in a new window]

  		FIG. 2. In situ hybridization analysis of WNT signaling pathway components in endometria and conceptuses from d 10 to 20 of pregnancy (P). In each column, representative photomicrographs of in situ hybridization results are presented in dark-field illumination. Mel, Melanocyte; S, stroma; Tr, trophectoderm. Scale bars, 100 µm.

 

Figure 3
View larger version (52K):
[in this window]
[in a new window]

  		FIG. 3. Immunohistochemical analysis of CTNNB1 and TCF7L2 in endometria and conceptuses from d 10 to 20 of pregnancy (P). Representative photomicrographs of results are presented, and sections were not counterstained. S, Stroma. Scale bars, 100 µm.

 
FZD6, FZD8, and GSK3B mRNAs were detected in the conceptus trophectoderm and uterine epithelia (Figs. 1Go and 2Go). The coreceptor LRP5 mRNA was observed in the conceptus trophectoderm and all endometrial cell types (Fig. 2Go), whereas the LRP6 mRNA was most abundant in the endometrial epithelia but at much lower abundance in trophectoderm and endometrial stroma (Fig. 2Go). GSK3B mRNA was detected in both the endometrium and conceptus (Fig. 2Go). DKK1 mRNA was very low or not detectable in both uteri or conceptus trophectoderm between d 10 and 14 but appeared on d 16 and was most abundant on d 18 and 20 in endometrial stroma. As expected, CDH1 mRNA was detected in endometrial epithelia and was particularly abundant in conceptus trophectoderm. The overall levels of CDH1 mRNA in endometrial LE declined from d 10 to 14 of pregnancy and then increased thereafter. CTNNB1 mRNA was detected in all uterine cell types but was most abundant in endometrial epithelia and conceptus trophectoderm. LEF1 mRNA was detected in endometrial epithelia but not in conceptus trophectoderm. Abundant MSX2 mRNA was detected in conceptus trophectoderm and in endometrial LE and sGE at much lower levels on d 18 and 20 of pregnancy. MYC mRNA was detected at low abundance in all uterine cell types and conceptus trophectoderm and was particularly abundant in trophoblast giant BNCs.

As expected from the in situ hybridization results, immunoreactive CTNNB1 protein was most abundant in the epithelia of the endometrium and conceptus (Fig. 2Go). In the endometrium, CTNNB1 protein decreased in the LE after d 10, whereas CTNNB1 protein was abundant in conceptus trophectoderm on d 18 and 20 of pregnancy. In the mononuclear cells of the trophectoderm, CTNNB1 protein was noticeably present at junctions between the cells. Furthermore, CTNNB1 protein was particularly abundant in the cytoplasm and nucleus of trophoblast giant BNCs and endodermal cells. TCF7L2 protein was abundant in nuclei of trophectoderm and endoderm cells of the conceptus. Collectively, these results suggest that WNT signaling via both canonical and noncanonical pathways is present and potentially active in cells of the endometrium and conceptus during the periimplantation period of pregnancy in sheep.

WNT7A stimulates trophectoderm cell proliferation and gene expression
We next focused on WNT7A, because it is a novel IFNT-stimulated gene expressed only in endometrial LE and ductal glands in the uterus of sheep and other mammals. WNT7A is hypothesized to act in a paracrine manner on conceptus trophectoderm based on results of the present study and a previous study from our laboratory (19).

oTr-1 cells, isolated from d 15 conceptuses, were found to express WNT2, WNT2B, WNT4, FZD6, FZD8, LRP5, LRP6, GSK3B, CTNNB1, CDH1, MSX2, MYC, and IFNT but not WNT7A or DKK1 at several different passages by RT-PCR (data not shown). Furthermore, the oTr-1 cells contained CTNNB1 and TCF7L2 proteins of the expected size in Western blot analyses (data not shown). As illustrated in Fig. 4Go, transfection analyses were used to study WNT7A signaling in oTr cells. In oTr cells transfected with a WNT7A overexpression vector, activity of the TOP-FLASH reporter, containing eight consensus LEF/TCF response elements, was stimulated (P < 0.001) 12-fold relative to cells transfected with the pCDNA3.1 empty vector. Although expression of dnTCF and Sfrp2 alone had no effect (P > 0.10) on basal activity of the TOP-FLASH reporter in the oTr cells, cotransfection of those inhibitors with WNT7A inhibited (P < 0.0001) reporter activity. Transfection of a constitutively active CTNNB1 (S33Y) and treatment with LiCl, a GSK3B inhibitor, increased (P < 0.0001) TOP-FLASH activity approximately 26- and 29-fold, respectively. Furthermore, transfection of WNT7A had no effect (P > 0.10) on a FOP-FLASH reporter containing mutated and inactivated LEF/TCF binding sites (data not shown). Collectively, these results indicate that the oTr cells have an intact canonical WNT signaling pathway that can be activated by WNT7A in vitro.


Figure 4
View larger version (7K):
[in this window]
[in a new window]

  		FIG. 4. WNT7A activation of the canonical signaling pathway in oTr cells. A trophectoderm cell line was established from a d 15 ovine conceptus and then transfected with a TCF-luciferase reporter (TOP-FLASH). Cells were cotransfected with pcDNA3.1 empty vector as a control or with WNT7A, dnTCF, Sfrp2, or S33Y CTNNB1 expression constructs. As a positive control, cells were treated with LiCl, a GSK3B inhibitor, for 24 h. Data are presented as relative luciferase units (RLU) with SE. These experiments were repeated at least three times. **, P < 0.0001.

 
As illustrated in Fig. 5Go, cell proliferation assays found that transfection of WNT7A increased (P < 0.01) oTr cell number by approximately 30%. Although Sfrp2 alone had no effect, Sfrp2 reduced (P < 0.05) WNT7A-induced cell proliferation. Next, the effects of WNT7A on oTr cell gene expression were studied by transfection and semiquantitative real-time PCR analysis (Fig. 6Go). The oTr cells were transfected with empty pcDNA3.1 vector, WNT7A, Sfrp2, or their combination. Transfection with WNT7A increased (P < 0.01) MSX2 and MYC mRNA levels in oTr cells by 2.1- and 1.6-fold, respectively, compared with cells transfected with vector alone. Although Sfrp2 alone had no effect, Sfrp2 inhibited (P < 0.01) WNT7A stimulation of MSX2 and MYC mRNAs in cotransfected oTr cells. However, neither WNT7A nor Sfrp2 or their combination affected (P > 0.10) expression of CST3 or CTSL genes in oTr cells. Collectively, these results indicate that WNT7A from the endometrial LE acts in a paracrine manner on conceptus trophectoderm to stimulate its cell proliferation and transcription of selected genes, such as MSX2 and MYC, via the canonical WNT signaling pathway.


Figure 5
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 5. WNT7A stimulates proliferation of oTr cells. A trophectoderm cell line was established from a d 15 ovine conceptus and then transfected with pcDNA3.1 empty vector as a control, WNT7A, Sfrp2, or their combination. Cell numbers were determined after 48 h, and data are expressed relative to pcDNA3.1 (100%). *, P < 0.05; **, P < 0.01.

 

Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 6. WNT7A increases expression of MSX2 and MYC but not CST3 and CTSL in oTr cells. A trophectoderm cell line was established from a d 15 ovine conceptus and then transfected with pcDNA3.1 empty vector as a control, WNT7A, Sfrp2, or their combination. Target mRNA was measured by real-time PCR. Data are expressed as normalized expression (40 – CT). *, P < 0.05.

 
Wnt5a stimulates trophectoderm cell migration
We next focused on WNT5A, because it and WNT11 are expressed constitutively in the endometrium during the periimplantation period of pregnancy and both primarily activate the noncanonical or planar cell polarity pathway involved in cell movement and migration during critical processes such as gastrulation in embryos (9). As illustrated in Fig. 7Go, treatment of oTr cells in serum- and insulin-free DMEM with 500 and 1000 ng/ml recombinant mouse Wnt5a considerably increased (P < 0.001) oTr cell migration. Treatment of oTr cells with Y27632, ROCK inhibitor, and JNK inhibitor did not (P > 0.10) affect the basal rate of migration in the absence of Wnt5a. However, the ROCK inhibitor Y27632, but not the JNK inhibitor, reduced (P < 0.0001) Wnt5a-stimulated oTr cell migration in a dose-dependent manner. Collectively, these results strongly support the idea that WNT5A, and perhaps WNT11, from the endometrium acts in a paracrine manner on conceptus trophectoderm to stimulate cell movement and migration via activation of the noncanonical WNT signaling pathway involving Rho kinase.


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 7. Wnt5A stimulates migration of oTr cells. The trophectoderm cell line established from a d 15 ovine conceptus was cultured in a transwell plate (n = three wells per treatment) and treated with recombinant mouse Wnt5a, Y27632 (ROCK inhibitor), JNK inhibitor, or their combination. Cells grown in serum- and insulin-containing DMEM served as a positive control. Cell migration was determined after 8 h of treatment and expressed as least-square means ± SE. Statistically significant (P < 0.001) differences in cell migration attributable to transfection with Wnt5A and inhibition of cell migration by Y27632 are noted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first comprehensive analysis of WNTs and their signaling pathway components in the ovine uterus and conceptus during the periimplantation period of pregnancy. Results of the present study indicate that WNTs using the canonical (WNT2, WNT2B, WNT4, and WNT7A) and noncanonical (WNT5A, WNT5B, and WNT11) signaling pathways are present in endometria and/or conceptuses during early pregnancy. Of particular note, several WNTs (WNT5A, WNT5B, and WNT11) are primarily of endometrial origin and hypothesized to act in a paracrine manner on the developing conceptus. Components of the WNT signaling pathway identified in endometria and/or conceptus trophectoderm include receptors (FZD6, FZD8, LRP5, and LRP6), signaling molecules (GSK3B and CTNNB1), and transcription factors (LEF1 and TCF7L2). Tulac et al. (17) found that WNTs using the canonical (WNT2, WNT3, WNT4, WNT7A, and WNT8B) and noncanonical (WNT5A) signaling pathways were expressed in endometria of women during the secretory phase of the menstrual cycle. In that study, WNT7A was detected only in uterine LE, but the spatial expression of the other WNTs was not reported. Few reports of expression of WNTs in uteri during early pregnancy in women or mice are available, but WNT4 and WNT5A are expressed predominantly in uterine stroma of mice during the estrous cycle, whereas WNT7A is expressed only in the LE (37). Collectively, available studies demonstrate that WNT7A is expressed only in endometrial LE of sheep, mice, and humans (17, 37), implicating the canonical WNT signaling pathway as a conserved regulator of endometrial function across mammals. In human uteri, WNT7A is present in the endometrial LE during both the proliferative and secretory phases of the menstrual cycle (17). In the present study, WNT7A mRNA was present on d 10, disappeared by d 12, reappeared on d 16, and increased to d 20. In the sheep, the temporal changes in WNT7A abundance correlate with loss of progesterone receptors from endometrial LE after d 10 of pregnancy (38) and secretion of IFNT by conceptus trophectoderm on d 16 (39). Indeed, progestins stimulate WNT7A in human endometrial epithelial cells (40), and our previous study determined that WNT7A was a novel IFNT-induced gene expressed only by the endometrial LE of the ovine uterus (19). The loss of WNT7A and CTNNB1 in the endometrial LE on d 12 and 14 may allow for synchronization of development of the preimplantation blastocyst and uterus as proposed in mice (41).

All essential components of the canonical WNT signaling pathway were identified in conceptus trophectoderm, including receptors (FZD6, FZD8, LRP5, and LRP6), signaling molecules (GSK3B and CTNNB1) and transcription factors (TCF7L2). Similarly, Pollheimer et al. (18) found that WNT ligands, FZD receptors, LRP6, and TCF7L1/TCF7L2 were in human placenta and various trophoblast cell lines. In mice, the canonical Wnt signaling pathway plays a central role in coordinating blastocyst-uterine interactions required for implantation (16). In the former study, canonical Wnt/Ctnnb1 signaling appeared first in circular smooth muscle cells of the myometrium on the antimesometrial side of the uterus early on d 4 of pregnancy and then became restricted to endometrial LE directly apposed to the blastocyst at the time of implantation. Activation of the canonical Wnt signaling pathway required the presence of the blastocyst and inhibition of Wnt/Ctnnb1 signaling interfered with implantation of the blastocyst (16). In the present study, LEF1 mRNA was observed primarily in endometrial epithelia during early pregnancy. Thus, WNT7A may act in an autocrine manner to regulate endometrial function, as well as in a paracrine manner to regulate periimplantation conceptus morphogenesis. Moreover, it is likely that the other WNTs identified in the endometrial stroma and/or conceptus (WNT2, WNT2B, and WNT4) regulate endometrial function and conceptus growth and differentiation as proposed in the mouse (42) and human (18). In fact, Wnt2 null mice have placentation defects, resulting in lower birth weights and perinatal lethality (43). The precise role of Wnt4 in placental development is not known, because Wnt4 homozygous null mice have a defect in differentiation of the fetal reproductive tract and suffer from perinatal lethality (44).

In the present study, the induction of WNT7A in the endometrial LE between d 14 and 16 of pregnancy was temporally correlated with elongation of the conceptus, production of IFNT, and the onset of trophoblast giant BNC differentiation. Indeed, our previous study determined that WNT7A was a novel IFNT-induced gene in the uterine LE (19). CTNNB1 and TCF7L2 proteins were particularly abundant in nuclei of trophoblast giant BNCs, suggesting that the canonical WNT signaling pathway is active in those cells of the conceptus. Indeed, transfection of WNT7A into mononuclear oTr cells stimulated the canonical WNT signaling pathway and cell proliferation and increased abundance of MSX2 and MYC mRNAs, which are genes up-regulated by the canonical WNT signaling pathway in other cell types (45, 46, 47, 48). MSX2, a homeobox transcription factor, is also expressed in the epithelial-mesenchymal cell layers of developing chorionic villi of human placentae (49). MYC, a transcription factor that stimulates genes required for proliferation, is also expressed in the human placenta in which it regulates cell proliferation (50). Homozygous null mice lacking Myc perish before 10.5 d of gestation and also exhibit reduced fertility in heterozygous female mice (51). Collectively, available evidence supports the idea that IFNT from the elongating conceptus acts in a paracrine manner to induce WNT7A in the endometrial LE, and, in turn, WNT7A acts in a paracrine manner on the conceptus to promote proliferation of the mononuclear cells and perhaps differentiation into trophoblast giant BNCs. Indeed, the canonical WNT signaling pathway promotes invasive human trophoblast differentiation in vitro (18), and CTNNB1/TCF has been implicated in trophoblast giant BNC differentiation in cattle (52). Although activation of the WNT signaling pathway was not demonstrated in that study, nuclear accumulation of CTNNB1 was observed during differentiation of bovine trophoblast cells into BNCs by endoreduplication (52). Thus, results from studies of human, cow, and sheep support the hypothesis that WNT/TCF signaling is involved in differentiation of multinucleated cells of the placenta, e.g. trophoblast giant BNCs or syncytiotrophoblast depending on the species.

In addition to WNT7A, parallels between human and sheep endometria were also apparent in the expression of DKK1, an inhibitor of WNT signaling. DKK1 interacts with LRP5/6 to inhibit WNT signaling by disrupting LRP5/6 binding to the WNT/FZD complex (53, 54). In the present study, DKK1 mRNA was detected specifically in the stratum compactum stroma of the endometrium beginning on d 16 and increased to d 20. In humans, DKK1 is regulated by progesterone in uterine endometrial stromal cells (55), induced in the secretory phase of the cycle (17), and increased during the window of implantation (56). Furthermore, Pollheimer et al. (18) found that DKK1 reduced migration, invasion, and proliferation of cytotrophoblasts from human placentae. Thus, the induction of DKK1 in the endometrial stroma of the ovine uterus is hypothesized to inhibit WNT signaling pathways in the trophoblast giant BNCs, and hence endometrial stroma, thereby preventing their migration into the stroma. The trophoblast giant BNCs arise from mononuclear trophectoderm cells that exit from the mitotic cycle, undergo subsequent endoreduplication, and then migrate and fuse with the endometrial LE and subsequently each other to form multinucleated syncytial plaques that line the chorionic villi of the cotyledonary portion of the placentomes (23). A hallmark of synepitheliochorial placentation in sheep is the lack of invasion of trophoblast giant BNCs and multinucleated syncytial plaques into the endometrial stroma of both caruncular and intercaruncular areas of the uterus (23, 24). Indeed, neither the BNCs nor multinucleated syncytia proliferate after their differentiation.

In addition to being required for canonical WNT signaling, CTNNB1 is a critical component of the cadherin cell adhesion complex that comprises a major cell adhesion system in epithelia (57). In the present study, CDH1 mRNA was abundant in the endometrial epithelia as well as conceptus trophectoderm, and CTNNB1 protein were localized both to the nucleus and at the periphery of endometrial epithelial cells and trophectoderm cells in d 18 and 20 conceptuses. Dynamic changes in the distribution of CDH1 and CTNNB1 in bovine conceptuses are hypothesized to have important roles in trophoblast giant BNC differentiation, including the rearrangement of cadherin-mediated cell adhesions during cell migration and the onset of endoreduplication, probably via nuclear transfer of CTNNB1 (52).

Results of the present studies also support the hypothesis that the noncanonical WNT or the planar cell polarity pathway also regulates conceptus development. WNT5A and WNT5B were expressed throughout early pregnancy in the endometrial stratum compactum stroma and at lower abundance in LE. WNT11 mRNA was also detected throughout early pregnancy but primarily in endometrial glands. Those WNTs appear to primarily activate the noncanonical or planar cell polarity pathway involved in cell movement and proliferation via activation of Rho-ROCK and JNK-JUN pathways (9). In mice, Wnt5a and Wnt11 were expressed after the morula-to-blastocyst transition, suggesting a candidate pathway for embryo-maternal signaling at implantation (58). However, null mutants of Wnt5a exhibit perinatal lethality (59), and Wnt11 mutants exhibit extensive embryonic lethality and perinatal lethality if they survive to term (60). In the present study, Wnt5a increased migration of oTr cells, which was inhibited by an inhibitor of ROCK but not an inhibitor of JNK. Thus, endometrial-derived WNT5A and WNT5B, as well as perhaps WNT11, are proposed to act in a paracrine manner on conceptus trophectoderm to stimulate movement and migration via the Rho-ROCK pathway. The Rho-ROCK pathway stimulates cell movement and migration by regulating the organization of actin filaments required to maintain cell adhesion during movement (61). In contrast to humans and rodents, blastocysts of domestic ruminants must elongate before implantation (21, 22). Trophoblast elongation involves cell migration and proliferation and is required for formation of the conceptus and developmentally regulated production of IFNT for pregnancy recognition (21, 62). In fact, blastocyst elongation is compromised in the uterine gland knockout ewe model, which lacks endometrial glands and has a reduced amount of LE and stroma (63). It is tempting to speculate that WNT11 may also be involved in conceptus elongation, because WNT11 mRNA was most abundant in endometrial glands in the present study. Recent evidence indicates that WNT5A and WNT11 regulate gastrulation in zebrafish by activation of Rho kinase (64). The process of gastrulation is very similar to blastocyst elongation in ruminants because both involve massive cell rearrangements, including convergence and extension movements that involve narrowing and lengthening of embryonic tissues as well as cell differentiation (21, 65). In sheep, gastrulation begins after d 9 and is completed by d 16 (65).

In summary, these results support the ideas that canonical and noncanonical WNT signaling pathways are conserved regulators of conceptus-endometrial interactions in mammals and regulate periimplantation ovine conceptus development. Collectively, available results support the following working hypotheses: 1) as blastocysts elongate to a filamentous form, WNT7A is induced in the endometrial LE between d 14 and 16 by IFNT from conceptus trophectoderm, and, in turn, endometrial WNT7A acts in a paracrine manner to stimulate proliferation, expression of TCF target genes such as MSX2 and MYC, and differentiation of mononuclear trophectoderm cells into trophoblast giant BNCs from d 14 to 16 of pregnancy; and 2) WNT5A, and perhaps WNT5B and WNT11, from the endometrium act in a paracrine manner to promote conceptus elongation via activation of the planar cell polarity pathway.


   Acknowledgments
 
We thank Mr. Kendrick LeBlanc and other members of our laboratory for assistance with animal husbandry and surgeries.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD38274 and 5 P30 ES09106.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 12, 2007

Abbreviations: BNC, Binucleate cell; CT, threshold cycle; dnTCF, dominant-negative human TCF7L2; FZD, frizzled receptor; IFNT, interferon {tau}; LE, luminal epithelium; oTr cells, ovine trophectoderm cells; ROCK, Rho kinase; SFRP, secreted frizzled-related protein; sGE, superficial glandular epithelium.

Received March 5, 2007.

Accepted for publication March 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Polakis P 2000 Wnt signaling and cancer. Genes Dev 14:1837–1851[Free Full Text]
  2. Dale TC 1998 Signal transduction by the Wnt family of ligands. Biochem J 329(Pt 2):209–223
  3. Wang Y, Macke JP, Abella BS, Andreasson K, Worley P, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J 1996 A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled. J Biol Chem 271:4468–4476[Abstract/Free Full Text]
  4. Liu X, Liu T, Slusarski DC, Yang-Snyder J, Malbon CC, Moon RT, Wang H 1999 Activation of a frizzled-2/ß-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via G{alpha}o and G{alpha}t. Proc Natl Acad Sci USA 96:14383–14388[Abstract/Free Full Text]
  5. Pinson KI, Brennan J, Monkley S, Avery BJ, Skarnes WC 2000 An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407:535–538[CrossRef][Medline]
  6. Finch PW, He X, Kelley MJ, Uren A, Schaudies RP, Popescu NC, Rudikoff S, Aaronson SA, Varmus HE, Rubin JS 1997 Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action. Proc Natl Acad Sci USA 94:6770–6775[Abstract/Free Full Text]
  7. Rattner A, Hsieh JC, Smallwood PM, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J 1997 A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Proc Natl Acad Sci USA 94:2859–2863[Abstract/Free Full Text]
  8. Nusse R 2001 Developmental biology. Making head or tail of Dickkopf. Nature 411:255–256[CrossRef][Medline]
  9. Logan CY, Nusse R 2004 The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20:781–810[CrossRef][Medline]
  10. Pandur P, Maurus D, Kuhl M 2002 Increasingly complex: new players enter the Wnt signaling network. Bioessays 24:881–884[CrossRef][Medline]
  11. Torres MA, Yang-Snyder JA, Purcell SM, DeMarais AA, McGrew LL, Moon RT 1996 Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development. J Cell Biol 133:1123–1137[Abstract/Free Full Text]
  12. Ishitani T, Kishida S, Hyodo-Miura J, Ueno N, Yasuda J, Waterman M, Shibuya H, Moon RT, Ninomiya-Tsuji J, Matsumoto K 2003 The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca2+ pathway to antagonize Wnt/ß-catenin signaling. Mol Cell Biol 23:131–139[Abstract/Free Full Text]
  13. Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ, Yang Y 2003 Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent ß-catenin degradation. J Cell Biol 162:899–908[Abstract/Free Full Text]
  14. Kobayashi A, Behringer RR 2003 Developmental genetics of the female reproductive tract in mammals. Nat Rev Genet 4:969–980[CrossRef][Medline]
  15. Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H 2004 Molecular cues to implantation. Endocr Rev 25:341–373[Abstract/Free Full Text]
  16. Mohamed OA, Jonnaert M, Labelle-Dumais C, Kuroda K, Clarke HJ, Dufort D 2005 Uterine Wnt/ß-catenin signaling is required for implantation. Proc Natl Acad Sci USA 102:8579–8584[Abstract/Free Full Text]
  17. Tulac S, Nayak NR, Kao LC, Van Waes M, Huang J, Lobo S, Germeyer A, Lessey BA, Taylor RN, Suchanek E, Giudice LC 2003 Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium. J Clin Endocrinol Metab 88:3860–3866[Abstract/Free Full Text]
  18. Pollheimer J, Loregger T, Sonderegger S, Saleh L, Bauer S, Bilban M, Czerwenka K, Husslein P, Knofler M 2006 Activation of the canonical wingless/T-cell factor signaling pathway promotes invasive differentiation of human trophoblast. Am J Pathol 168:1134–1147[Abstract/Free Full Text]
  19. Kim S, Choi Y, Bazer FW, Spencer TE 2003 Identification of genes in the ovine endometrium regulated by interferon tau independent of signal transducer and activator of transcription 1. Endocrinology 144:5203–5214[Abstract/Free Full Text]
  20. Spencer TE, Bazer FW 2002 Biology of progesterone action during pregnancy recognition and maintenance of pregnancy. Front Biosci 7:d1879–d1898
  21. Guillomot M 1995 Cellular interactions during implantation in domestic ruminants. J Reprod Fertil Suppl 49:39–51[Medline]
  22. Spencer TE, Johnson GA, Bazer FW, Burghardt RC 2004 Implantation mechanisms: insights from the sheep. Reproduction 128:657–668[Abstract/Free Full Text]
  23. Wooding FB 1984 Role of binucleate cells in fetomaternal cell fusion at implantation in the sheep. Am J Anat 170:233–250[CrossRef][Medline]
  24. Wooding FB 1992 Current topic: the synepitheliochorial placenta of ruminants: binucleate cell fusions and hormone production. Placenta 13:101–113[Medline]
  25. Hayashi K, Spencer TE 2006 WNT pathways in the neonatal ovine uterus: potential specification of endometrial gland morphogenesis by SFRP2. Biol Reprod 74:721–733[Abstract/Free Full Text]
  26. Rozen S, Skaletsky H 2000 Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386[Medline]
  27. Spencer TE, Stagg AG, Joyce MM, Jenster G, Wood CG, Bazer FW, Wiley AA, Bartol FF 1999 Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model. Endocrinology 140:4070–4080[Abstract/Free Full Text]
  28. Taylor KM, Gray CA, Joyce MM, Stewart MD, Bazer FW, Spencer TE 2000 Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin. Biol Reprod 63:1192–1204[Abstract/Free Full Text]
  29. Johnson GA, Bazer FW, Jaeger LA, Ka H, Garlow JE, Pfarrer C, Spencer TE, Burghardt RC 2001 Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep. Biol Reprod 65:820–828[Abstract/Free Full Text]
  30. Dunlap KA, Palmarini M, Varela M, Burghardt RC, Hayashi K, Farmer JL, Spencer TE 2006 Endogenous retroviruses regulate periimplantation placental growth and differentiation. Proc Natl Acad Sci USA 103:14390–14395[Abstract/Free Full Text]
  31. Veeman MT, Slusarski DC, Kaykas A, Louie SH, Moon RT 2003 Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol 13:680–685[CrossRef][Medline]
  32. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW 1997 Activation of ß-catenin-Tcf signaling in colon cancer by mutations in ß-catenin or APC. Science 275:1787–1790[Abstract/Free Full Text]
  33. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H 1997 Constitutive transcriptional activation by a ß-catenin-Tcf complex in APC–/– colon carcinoma. Science 275:1784–1787[Abstract/Free Full Text]
  34. Hou X, Tan Y, Li M, Dey SK, Das SK 2004 Canonical Wnt signaling is critical to estrogen-mediated uterine growth. Mol Endocrinol 18:3035–3049[Abstract/Free Full Text]
  35. Raspotnig G, Fauler G, Jantscher A, Windischhofer W, Schachl K, Leis HJ 1999 Colorimetric determination of cell numbers by Janus green staining. Anal Biochem 275:74–83[CrossRef][Medline]
  36. Hayashi K, Carpenter KD, Spencer TE 2004 Neonatal estrogen exposure disrupts uterine development in the postnatal sheep. Endocrinology 145:3247–3257[Abstract/Free Full Text]
  37. Miller C, Pavlova A, Sassoon DA 1998 Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle. Mech Dev 76:91–99[CrossRef][Medline]
  38. Spencer TE, Bazer FW 1995 Temporal and spatial alterations in uterine estrogen receptor and progesterone receptor gene expression during the estrous cycle and early pregnancy in the ewe. Biol Reprod 53:1527–1543[Abstract]
  39. Ashworth CJ, Bazer FW 1989 Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone. Biol Reprod 40:425–433[Abstract]
  40. Oehler MK, MacKenzie IZ, Wallwiener D, Bicknell R, Rees MC 2002 Wnt-7a is upregulated by norethisterone in human endometrial epithelial cells: a possible mechanism by which progestogens reduce the risk of estrogen-induced endometrial neoplasia. Cancer Lett 186:75–81[CrossRef][Medline]
  41. Li J, Zhang JV, Cao YJ, Zhou JX, Liu WM, Fan XJ, Duan EK 2005 Inhibition of the ß-catenin signaling pathway in blastocyst and uterus during the window of implantation in mice. Biol Reprod 72:700–706[Abstract/Free Full Text]
  42. Kemp C, Willems E, Abdo S, Lambiv L, Leyns L 2005 Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development. Dev Dyn 233:1064–1075[CrossRef][Medline]
  43. Monkley SJ, Delaney SJ, Pennisi DJ, Christiansen JH, Wainwright BJ 1996 Targeted disruption of the Wnt2 gene results in placentation defects. Development 122:3343–3353[Abstract]
  44. Stark K, Vainio S, Vassileva G, McMahon AP 1994 Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature 372:679–683[CrossRef][Medline]
  45. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW 1998 Identification of c-MYC as a target of the APC pathway. Science 281:1509–1512[Abstract/Free Full Text]
  46. Willert J, Epping M, Pollack JR, Brown PO, Nusse R 2002 A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC Dev Biol 2:8[CrossRef][Medline]
  47. Hussein SM, Duff EK, Sirard C 2003 Smad4 and ß-catenin co-activators functionally interact with lymphoid-enhancing factor to regulate graded expression of Msx2. J Biol Chem 278:48805–48814[Abstract/Free Full Text]
  48. Shu W, Guttentag S, Wang Z, Andl T, Ballard P, Lu MM, Piccolo S, Birchmeier W, Whitsett JA, Millar SE, Morrisey EE 2005 Wnt/ß-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. Dev Biol 283:226–239[CrossRef][Medline]
  49. Quinn LM, Latham SE, Kalionis B 2000 The homeobox genes MSX2 and MOX2 are candidates for regulating epithelial-mesenchymal cell interactions in the human placenta. Placenta 21 (Suppl A):S50–S54
  50. Pfeifer-Ohlsson S, Goustin AS, Rydnert J, Wahlstrom T, Bjersing L, Stehelin D, Ohlsson R 1984 Spatial and temporal pattern of cellular myc oncogene expression in developing human placenta: implications for embryonic cell proliferation. Cell 38:585–596[CrossRef][Medline]
  51. Davis AC, Wims M, Spotts GD, Hann SR, Bradley A 1993 A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. Genes Dev 7:671–682[Abstract/Free Full Text]
  52. Nakano H, Shimada A, Imai K, Takahashi T, Hashizume K 2005 The cytoplasmic expression of E-cadherin and ß-catenin in bovine trophoblasts during binucleate cell differentiation. Placenta 26:393–401[CrossRef][Medline]
  53. Fedi P, Bafico A, Nieto Soria A, Burgess WH, Miki T, Bottaro DP, Kraus MH, Aaronson SA 1999 Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. J Biol Chem 274:19465–19472[Abstract/Free Full Text]
  54. Bafico A, Liu G, Yaniv A, Gazit A, Aaronson SA 2001 Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat Cell Biol 3:683–686[CrossRef][Medline]
  55. Tulac S, Overgaard MT, Hamilton AE, Jumbe NL, Suchanek E, Giudice LC 2006 Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells. J Clin Endocrinol Metab 91:1453–1461[Abstract/Free Full Text]
  56. Kao LC, Tulac S, Lobo S, Imani B, Yang JP, Germeyer A, Osteen K, Taylor RN, Lessey BA, Giudice LC 2002 Global gene profiling in human endometrium during the window of implantation. Endocrinology 143:2119–2138[Abstract/Free Full Text]
  57. Takeichi M 1995 Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7:619–627[CrossRef][Medline]
  58. Mohamed OA, Dufort D, Clarke HJ 2004 Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation. Biol Reprod 71:417–424[Abstract/Free Full Text]
  59. Yamaguchi TP, Bradley A, McMahon AP, Jones S 1999 A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 126:1211–1223[Abstract]
  60. Majumdar A, Vainio S, Kispert A, McMahon J, McMahon AP 2003 Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development. Development 130:3175–3185[Abstract/Free Full Text]
  61. Nobes CD, Hall A 1999 Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 144:1235–1244[Abstract/Free Full Text]
  62. Guillomot M, Michel C, Gaye P, Charlier N, Trojan J, Martal J 1990 Cellular localization of an embryonic interferon, ovine trophoblastin and its mRNA in sheep embryos during early pregnancy. Biol Cell 68:205–211[CrossRef][Medline]
  63. Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE 2002 Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation. Reproduction 124:289–300[Abstract]
  64. Zhu S, Liu L, Korzh V, Gong Z, Low BC 2006 RhoA acts downstream of Wnt5 and Wnt11 to regulate convergence and extension movements by involving effectors Rho kinase and Diaphanous: use of zebrafish as an in vivo model for GTPase signaling. Cell Signal 18:359–372[CrossRef][Medline]
  65. Guillomot M, Turbe A, Hue I, Renard JP 2004 Staging of ovine embryos and expression of the T-box genes Brachyury and Eomesodermin around gastrulation. Reproduction 127:491–501[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
K. Hayashi, D. W. Erikson, S. A. Tilford, B. M. Bany, J. A. Maclean II, E. B. Rucker III, G. A. Johnson, and T. E. Spencer
Wnt Genes in the Mouse Uterus: Potential Regulation of Implantation
Biol Reprod, May 1, 2009; 80(5): 989 - 1000.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. Kim, G. Song, H. Gao, J. L. Farmer, M. C. Satterfield, R. C. Burghardt, G. Wu, G. A. Johnson, T. E. Spencer, and F. W. Bazer
Insulin-Like Growth Factor II Activates Phosphatidylinositol 3-Kinase-Protooncogenic Protein Kinase 1 and Mitogen-Activated Protein Kinase Cell Signaling Pathways, and Stimulates Migration of Ovine Trophectoderm Cells
Endocrinology, June 1, 2008; 149(6): 3085 - 3094.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
S. Ray, F. Xu, H. Wang, and S. K. Das
Cooperative Control via Lymphoid Enhancer Factor 1/T Cell Factor 3 and Estrogen Receptor-{alpha} for Uterine Gene Regulation by Estrogen
Mol. Endocrinol., May 1, 2008; 22(5): 1125 - 1140.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
T. E Spencer, O. Sandra, and E. Wolf
Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches
Reproduction, February 1, 2008; 135(2): 165 - 179.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
J. L. Farmer, R. C. Burghardt, F. D. Jousan, P. J. Hansen, F. W. Bazer, and T. E. Spencer
Galectin 15 (LGALS15) functions in trophectoderm migration and attachment
FASEB J, February 1, 2008; 22(2): 548 - 560.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hayashi, K.
Right arrow Articles by Spencer, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hayashi, K.
Right arrow Articles by Spencer, T. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals