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BOR - Papers in Press, published online ahead of print January 11, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.049718
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BIOLOGY OF REPRODUCTION 74, 721–733 (2006)
DOI: 10.1095/biolreprod.105.049718
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

WNT Pathways in the Neonatal Ovine Uterus: Potential Specification of Endometrial Gland Morphogenesis by SFRP21

Kanako Hayashi , and Thomas E. Spencer 2 

Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471

ABSTRACT

Endometrial glands are critical for uterine function and develop between birth (Postnatal Day [P] 0) and P56 in the neonatal ewe. Endometrial gland morphogenesis or adenogenesis involves the site-specific budding differentiation of the glandular epithelium from the luminal epithelium followed by their coiling/branching development within the stroma of the intercaruncular areas of the endometrium. To determine whether WNT signaling regulates endometrial adenogenesis, the WNT signaling system was studied in the neonatal ovine uterus. WNT5A, WNT7A, and WNT11 were expressed in the uterine epithelia, whereas WNT2B was in the stroma. The WNT receptors FZD2 and FZD6 and coreceptor LRP6 were detected in all uterine cells, and FZD6 was particularly abundant in the endometrial epithelia. Secreted FZD-related protein-2 (SFRP2), a WNT antagonist, was not detected in the P0 uterus, but was abundant in the aglandular caruncular areas of the endometrium between P7 and P56. Exposure of ewes to estrogens during critical developmental periods inhibits or retards endometrial adenogenesis. Estrogen-induced disruption of endometrial adenogenesis was associated with reduction or ablation of WNT2B, WNT7A, and WNT11, and with an increase in WNT2 and SFRP2 mRNA, depending on exposure period. Collectively, results implicate the canonical and noncanonical WNT pathways in regulation of postnatal ovine uterine development and endometrial adenogenesis. Expression of SFRP2 in aglandular caruncular areas may inhibit the WNT signaling pathway, thereby concentrating WNT signaling and restricting endometrial adenogenesis in the intercaruncular areas of the uterus. Further, estrogen-induced inhibition of adenogenesis may be mediated by a reduction in WNT signaling caused by aberrant induction of SFRP2 and loss of several critical WNTs.

development, developmental biology, endometrium, estradiol, kinases, SFRP, sheep, signal transduction, uterus, WNT

INTRODUCTION

Although histogenesis of the uterus is initiated in the fetus, uterine development is not completed until after birth in humans, domestic animals, and laboratory rodents [1, 2]. A major developmental event in the neonatal uterus is the differentiation and development of the endometrial glands, or adenogenesis [3, 4]. Alteration or ablation of endometrial glands and/or their secretory products compromises survival and growth of the conceptus (embryo/fetus and associated extraembryonic membranes) in the mouse, rat, pig, cow, and sheep [1, 5, 6]. In humans, the secretory products of endometrial glands appear to be a primary source of nutrition for conceptus growth during the first trimester [7]. In sheep, uterine development after birth involves differentiation of the endometrial glandular epithelium (GE) from the luminal epithelium (LE), specification and development of the intercaruncular endometrial stroma, development of endometrial folds, and, to a lesser extent, growth of endometrial caruncular areas and the myometrium [8, 9]. The caruncular areas of the endometrium, which are the sites of superficial implantation and placentation for formation of placentomes, do not contain any glands. Adult uterine gland knockout (UGKO) ewes are infertile and exhibit recurrent early pregnancy loss [4, 10].

Exposure of neonates to estrogen or other estrogen receptor alpha (ESR1) agonists during critical developmental periods induces a uterotrophic response and either inhibits or potentiates adenogenesis in the rat and pig, respectively [11–14]. In sheep, exposure of the neonatal ewe to 17ß-estradiol valerate (EV) from birth during the infantile phase (Postnatal Day [P] 1–P14) reduced uterine growth and completely ablated endometrial adenogenesis [15, 16]. Transient exposure of neonatal ewes to 17ß-estradiol benzoate (EB) during the tubulogenic (P14–P28) and coiling/branching (P42–P56) phases of uterine development reduced endometrial adenogenesis and altered growth factor networks, leading to long-term alterations in uterine gene expression and structure [17]. The effects of inappropriate exposure to ovarian steroid hormones on uteri of sheep indicate that postnatal uterine development contains critical windows that are exquisitely sensitive to the disruptive effects of endocrine disruptors and have permanent effects on uterine structure and function.

WNT genes are homologous to the Drosophila segment polarity gene wingless (wg). In humans and mice, the WNT family encodes a group of 19 highly conserved secreted glycoproteins that regulate cell and tissue growth and differentiation during gland morphogenesis in other epitheliomesenchymal organs, such as the mammary gland and kidney [18–20]. To be effective in autocrine or paracrine signaling, WNTs associate with their extracellular surface receptors, frizzled (FZD), to mediate intracellular signal transduction pathways [21]. The 10 FZDs are a family of seven transmembrane G protein-coupled receptors that possess an extracellular cysteine-rich domain for WNT binding [22, 23]. Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) serve as coreceptors with FZD [24]. Another family of proteins, termed secreted FZD-related proteins (SFRPs) produced by six different genes, exerts inhibitory effects on WNT signaling by competing with WNT ligands for the FZD receptor and forming a nonfunctional complex with FZDs in a dominant-negative manner [25, 26]. The four Dickkopf (DKK) genes encode secreted proteins that bind the LRP coreceptor and also are antagonists of the WNT signaling pathway [27]. In the canonical WNT/beta-catenin (CTNNB1)/TCF signaling pathway, FZD receptors transduce a signal to several intracellular proteins that include disheveled (DVL), glycogen synthase kinase 3 beta (GSK3B), axin, adenomatous polyposis coli, and the transcriptional regulator CTNNB1 [28, 29]. Nuclear CTNNB1 interacts with transcription factors, most notably members of the transcription factor 7 (T-cell specific, HMG-box) (TCF7/LEF) family, to regulate transcription of genes such as JUN (c-Jun) [30], LEF1/TCF7 [31], MET (c-Met) [32], and MSX2 (msh homeobox homolog 2) [33, 34]. The noncanonical or planar cell polarity pathways, activated by WNT5A and WNT11, mediate cell polarity, cell movements during gastrulation, and other processes by signal transduction through DVL, leading to a modification of the actin cytoskeleton by small GTPases of the Rho family, such as Rho, Rac, and Cdc42 [35–38]. Rac activation stimulates c-Jun N-terminal kinase (JNK) activity [39–41]. JNK activation plays essential roles in organogenesis by regulating cell survival, apoptosis, and proliferation [42].

In mice, three members of the WNT gene family (Wnt4, Wnt5a, and Wnt7a) have been identified and studied in the female reproductive tract [43]. Null mutation of these genes or disruption of their expression by endocrine disruptors alters prenatal and postnatal development of the Müllerian duct (see [2, 44–46]). Wnt4-null mutants fail to form Müllerian ducts and die at birth because of numerous defects [47]. Analyses of the Wnt5a and Wnt7a mutants demonstrate the requirement of both genes in fetal uterine development and postnatal endometrial adenogenesis in mice [48, 49]. The WNT system has not been systematically investigated in the neonatal uterus of any species. Our working hypothesis is that WNTs expressed in the uterus are conserved critical regulators of uterine development, specifically epithelial growth and morphogenesis, via autocrine and/or paracrine actions mediated by both canonical and noncanonical WNT signaling pathways. As a first step in testing this hypothesis, we determined temporal and spatial alterations in the WNT system during postnatal ovine uterine development as well as effects of estrogen-induced disruption on the WNT system. The results reveal that the canonical and noncanonical WNT signaling pathways are present in the developing postnatal ovine uterus and that specification of gland development to the intercaruncular areas of the endometrium may be attributable to abundant SFRP2 expression in the aglandular caruncular areas on the endometrium. The potential importance of WNT signaling in uterine development is the finding that specific WNTs are inhibited and SFRP2 is increased by estrogen exposure that is associated with ablation or reduction of endometrial adenogenesis.

MATERIALS AND METHODS

Animals and Experimental Design

All experiments and surgical procedures were in accordance with the Guide for the Care and Use of Agriculture Animals in Agricultural Research and Teaching and approved by the University Laboratory Animal Care Committee of Texas A&M University. Ewes were obtained from the Texas A&M University Physiology Field Laboratory and maintained according to normal animal husbandry practices.

Study 1. Crossbred Suffolk ewes were mated to Suffolk rams between September and November of 2001. Pregnant ewes were maintained according to normal husbandry practices. Ewes included in the following experiments were born between January and May of 2002. Ewes (n = 45) were assigned randomly at birth or P0 to be hysterectomized on P0 (n = 6), P7 (n = 4), P14 (n = 5), P21 (n = 5), P28 (n = 5), P42 (n = 5), or P56 (n = 5). The entire reproductive tract (uterus and ovary) was excised, and the uterus was trimmed free of the broad ligament, oviduct, and cervix. Sections from the middle of each uterine horn (~1 cm) were fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% (v/v) ethanol and then embedded in Paraplast Plus (Oxford Labware). The remainder of the uterine horn was frozen in liquid nitrogen and stored at –80°C for RNA extraction.

Study 2. Crossbred Suffolk ewes were mated to Suffolk rams in October and November of 2002. Ewes were born in February and March of 2003. Ewes (n = 5 per treatment) were assigned randomly at birth (P 0) to receive daily i.m. injections from P0 to P13 of: 1) corn oil vehicle as a control (CX); 2) EV (50 µg/kg body weight [BW]) in corn oil. This dose of EV disrupts uterine development and endometrial adenogenesis in the neonatal ewe [15]. Ewes were weighed on P7, and the EV dose adjusted accordingly. On P14, the entire reproductive tract (uterus and ovary) was excised, and the uterus was trimmed free of the broad ligament, oviduct, and cervix. Sections (~1 cm) from the mid portion of the uterine horn were fixed in fresh 4% (w/v) paraformaldehyde in PBS (pH 7.2) at room temperature for 24 h and processed for histology. The remainder of the uterine horn was frozen in liquid nitrogen and stored at –80°C for RNA extraction.

Study 3. Crossbred Suffolk ewes were mated to Suffolk rams between September and November of 2002. Ewes included in the following experiments were born between January and May of 2003. As described previously [17], ewes (n = 20) were assigned randomly at birth or P0 to receive daily i.m. injections from P14 to P27 (period 1) or from P42 to P55 (period 2) of: 1) CX, or 2) EB (Sigma Chemical Co.) in corn oil at a dose of 10 µg/kg BW (EB-10). Ewes were weighed every 7 days, and the EB dose adjusted accordingly. To determine the immediate effects of estrogen exposure on reproductive tract development, ewes were weighed and surgically hemiovariohysterectomized 24 h after the last treatment with EB on either P28 (period 1) or P56 (period 2). Briefly, the right ovarian pedicle and the right uterine horn were ligated with suture immediately above the intercornual ligament, and the anterior portion of the right uterine horn above the ligature was removed. Two pieces (~1 cm) of the middle region of the uterine horn were fixed in fresh 4% (w/v) paraformaldehyde in PBS (pH 7.2) at room temperature for 24 h and processed for histology. The remainder of the uterine horn was frozen in liquid nitrogen and stored at –80°C for RNA extraction. On P112, all ewes were weighed, and the entire reproductive tract was removed. The uterus was obtained and trimmed free of the broad ligament, oviduct and cervix. Sections (~1 cm) from the mid portion of the uterine horn were fixed in 4% (w/v) paraformaldehyde fixative, and the remainder of the uterus was frozen in liquid nitrogen and stored at –80°C.

RT-PCR Analysis

Expression of components of the WNT signaling pathways was studied by RT-PCR as described previously [16, 17, 50, 51]. Primers for each component were derived from conserved sequences of human and bovine genes using Primer 3 [52]. A partial ovine cDNA of 240–600 bp was cloned by RT-PCR using total RNA isolated from either the neonatal uterus or ovary or the adult endometrium. Primer and annealing temperatures used for PCR are summarized in Table 1. The amplified PCR products were subcloned into the pCRII cloning vector using a T/A Cloning Kit (Invitrogen Life Technologies) and sequenced in both directions using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems) to confirm identity.


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  		TABLE 1. Sequences of primers for RT-PCR.

In Situ Hybridization

In situ hybridization analysis of ovine uteri were conducted using methods described previously [53]. 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 [35S-{alpha}]UTP. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Kodak), stored at 4°C for 4–60 days, and developed in Kodak D-19 developer. Slides were then counterstained with Gill's modified hematoxylin (Stat Lab), dehydrated through a graded series of alcohol to xylene, and protected with a coverslip.

Immunohistochemistry

Immunolocalization of CTNNB1, TCF7L2, p-JNK, JUN, and p-JUN proteins was performed in cross-sections (5 µm) of the paraffin-embedded uterus from each ewe using specific antibodies and a Vectastain ABC Kit with mouse IgG (PK-6102) or rabbit IgG (PK-6101). Mouse monoclonal antibody to phosphorylated mouse CTNNB1 (610153; BD Biosciences), mouse monoclonal anti-human TCF7L2 antibody (05–511; Upstate), rabbit antiserum against human JUN (06–225; Upstate), a rabbit anti-phospho-JNK antibody (07–175; Upstate), and phospho-JUN (06–659; Upstate) were used for immunohistochemistry. The working antibody concentrations employed for immunohistochemistry were 1.0 µg/ml for CTNNB1, 0.5 µg/ml TCF7L2, 2.0 µg/ml for p-JNK, 1:2000 for JUN, and 1:200 for phospho-JUN. Negative controls were performed in which the primary antibody was substituted with the same concentration of normal mouse IgG, rabbit IgG, or rabbit serum from Sigma Chemical Company. Antigen retrieval using a boiling citrate buffer was performed for all antibodies as described previously [9]. Multiple tissue sections from each ewe were processed as sets within an experiment. Sections were not counterstained before affixing the coverslip.

Photomicroscopy

As described previously [54], relative amounts of mRNA and immunoreactive protein expression from in situ hybridization and immunohistochemistry analyses, respectively, were assessed visually in uterine sections (n = 2 per horn) from each ewe in each experiment by two independent observers and scored as follows: absent (–; i.e., no staining above sense or IgG control), weak (+), moderate (++), or strong (+++). The scores from the two observers were averaged. If histologically discernable, intercaruncular endometrial tissues (including LE, stroma, and GE) and caruncular endometrial tissues (including LE and stroma) were scored. Images of representative fields of sections hybridized with antisense or sense cRNAs were recorded under brightfield or darkfield illumination with a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc.) fitted with a Nikon DXM1200 digital camera using constant image acquisition parameters to ensure accurate comparison. Representative photomicrographs of protein immunolocalization were recorded using a Nikon Eclipse 1000 photomicroscope fitted with a Nikon DXM1200 digital camera.

RESULTS

Cloning of Partial cDNAs for Ovine WNT Signaling

RT-PCR was conducted using primers generated from conserved regions of the 19 human and bovine WNT genes and 10 human and bovine FZD genes and total RNA isolated from neonatal ovine uteri (P0 to P56). Positive control RNA was isolated from adult ovine endometrium as well as from ovaries from neonatal and adult ewes. Partial cDNAs of the correct predicted size (Table 1) were cloned and sequenced in both directions to confirm identity (data not shown). RT-PCR analyses found that only 6 of the 19 WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT7A, and WNT11), and only 3 of the 10 FZD genes (FZD2, FZD6, and FZD8) are expressed in the neonatal ovine uterus (data not shown). The partial ovine FZD2 cDNA shared significant homology (87%–94%) with human FZD1, FZD2, FZD7, and FZD10. Three of the five SFRP genes (SFRP1, SFRP2, and SFRP4) were expressed in the neonatal ovine uterus. One of the four DKK genes (DKK1) was detected by RT-PCR. Expression of LRP5, LRP6, GSK3B, CTNNB1, CDH1, MSX1, and MSX2 mRNAs was detected by RT-PCR in the neonatal ovine uterus.

Localization of WNT System in the Neonatal Ovine Uterus (Study 1)

In situ hybridization and/or immunohistochemistry analyses were conducted to localize expression of WNT system components in the neonatal ovine uterus. Representative photomicrographs are presented in Figures 1, 2 and 3, and expression patterns are summarized in Table 2. The endometrium of many ewes contained melanocytes whose dark pigment turns white under darkfield illumination; however, the melanocytes do not express any of the WNT signaling pathway components. WNT2 mRNA abundance was below the limit of detection by in situ hybridization analysis (Fig. 1). In contrast, WNT2B mRNA was located only in the intercaruncular stroma surrounding the developing GE from P14 to P56, and was not observed on P0 or P7. WNT4 mRNA was detected at low abundance in all cell types in the neonatal ovine uterus. WNT5A mRNA was detected predominantly in the LE on P0. WNT5A mRNA was detected predominantly in the GE and at a lower abundance in the LE and stroma from P7 to P56. WNT7A mRNA was detected specifically in the LE on P0 and only in the LE and superficial ductal GE (sGE) thereafter. WNT11 mRNA was observed only in the LE on P0 and declined in the LE and sGE after P0.


Figure 1
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  		FIG. 1. In situ hybridization analysis of WNT system components in the neonatal ovine uterus. In each panel portion, representative photomicrographs of in situ hybridization results are presented in darkfield illumination. The black melanocytes (Mel) in the uterus appear white under darkfield illumination. Car, Caruncular endometrium; GE, glandular epithelium; LE, luminal epithelium; M, myometrium; S, stroma. Bar = 100 µm.


Figure 2
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  		FIG. 2. In situ hybridization analysis of SFRP2 mRNA in the neonatal ovine uterus. In each panel portion, representative photomicrographs of in situ hybridization results are presented in darkfield illumination. The black melanocytes (Mel) in the uterus appear white under darkfield illumination. Car, Caruncular endometrium; GE, glandular epithelium; LE, luminal epithelium; M, myometrium; S, stroma. Bar = 100 µm.


Figure 3
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  		FIG. 3. Immunohistochemical analysis of WNT system components in the neonatal ovine uterus. In each panel portion, representative photomicrographs of immunohistochemical localization results are presented. Note the black melanocytes (Mel) in the uterus. Car, Caruncular endometrium; GE, glandular epithelium; LE, luminal epithelium; M, myometrium; S, stroma. Bar = 100 µm.


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  		TABLE 2. WNT signaling components in the neonatal ovine uterus: location in the endometrium and effects of estrogen exposure.

In situ hybridization analyses revealed that FZD2 mRNA was present in all uterine cell types, but was most abundant in the endometrial stroma and the myometrium. FZD6 mRNA was also present in all endometrial cell types, but was most abundant in the endometrial LE and GE. FZD8 mRNA was below the limit of detection by in situ hybridization (data not shown). LRP6 mRNA was present in all uterine cell types. LRP5 and DKK1 mRNAs were below the limit of detection by in situ hybridization (data not shown). SFRP1 mRNA was detected in low abundance in the stroma. SFRP2 mRNA was not detected on P0 (Figs. 1 and 2). Once endometrial gland development was initiated in the intercaruncular regions, SFRP2 mRNA was detected in the stroma of the aglandular caruncular endometrium by P7 and remained abundant in those cells thereafter. In the intercaruncular areas of the endometrium of P56 ewes, SFRP2 mRNA was expressed in the stroma between the tips of the developing GE and the inner circular layer of the myometrium. SFRP4 mRNA was below the limit of detection by in situ hybridization (data not shown).

As expected, CHD1 mRNA was expressed only in the endometrial LE and GE (Fig. 1). GSK3B and CTNNB1 mRNAs were present predominantly in the LE on P0 and most abundantly in the LE and GE of the endometrium thereafter to P56. Low levels of GSK3B and CTNNB1 mRNAs were also observed in endometrial stroma. MSX1 and MSX2 mRNAs were abundantly expressed in the endometrial LE and GE, but not in any other uterine cells.

Immunoreactive CTNNB1 protein was abundantly observed in the LE and GE and was present at lower levels in the stroma and the myometrium (Fig. 3). Abundant levels of immunoreactive TCF7L2 were observed in nuclei of the endometrial LE and GE, and TCF7L2 protein was also present at much lower abundance in the stroma. Immunoreactive activated JNK (p-JNK) was detected in all endometrial cell types but was most abundant in the endometrial LE and GE. Immunoreactive JUN and activated JUN (p-JUN) protein was observed in the nuclei of all endometrial cells.

Effects of Exposure to Estrogen on the Uterine WNT System During the Infantile Phase (P0–P14)

In study 2, exposure of neonatal ewes to EV during the infantile phase (P0–P14) completely inhibited endometrial gland genesis (Fig. 4). Effects of EV exposure during this phase on uterine histoarchitecture and several intrinsic growth factor systems has been published (see [15, 16]). Representative photomicrographs are presented in Figures 4 and 5, and effects of EV treatment on uterine mRNAs and protein are summarized in Table 2. EV exposure decreased WNT7A mRNA in the endometrial LE and completely ablated WNT2B and WNT11 mRNA in the stroma and LE, respectively. WNT5A mRNA was observed in the stroma at low levels in the uteri of EV ewes. Although not detected in the uteri of control ewes, WNT2 mRNA was induced in the endometrial stroma of ewes exposed to EV. Expression of WNT4, FZD2, FZD6, LRP6, and SFRP1 was not affected by EV exposure. In contrast, EV treatment increased SFRP2 mRNA in the stroma. MSX1 mRNA was decreased in the endometrial LE of uteri from EV-exposed ewes, whereas MSX2 mRNA was increased in the LE of EV-exposed ewes (Fig. 4).


Figure 4
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  		FIG. 4. Effects of EV exposure from P0 to P14 on the uterine WNT system. In each panel portion, representative photomicrographs of in situ hybridization results are presented in darkfield illumination. The black melanocytes in the uterus appear white under darkfield illumination. Car, Caruncular endometrium; GE, glandular epithelium; LE, luminal epithelium; M, myometrium; S, stroma. Bar = 100 µm.


Figure 5
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  		FIG. 5. Effects of EV exposure from P0 to P14 on the uterine WNT system. In each panel portion, representative photomicrographs of immunostaining results are presented. GE, Glandular epithelium; LE, luminal epithelium; S, stroma. Bar = 100 µm.

Exposure to EV did not affect GSK3B or CHD1 mRNA abundance or CTNNB1 mRNA or protein (Figs. 4 and 5). In contrast, TCF7L2 protein was substantially reduced in the LE, but not in the stroma, by EV treatment. Immunoreactive p-JNK, JUN, and p-JUN expression was not affected by EV exposure.

Effects of Exposure to Estrogen on the Uterine WNT System During the Tubulogenic (P14–28) and Coiling/Branching (P42–P56) Phases

In study 3, transient exposure of neonatal ewes to EB during the tubulogenic (P14–P27) or coiling/branching (P42–P55) phases of uterine development decreased endometrial gland development (Fig. 6). The effects of EB exposure during these phases on uterine histoarchitecture have been published (see [17]). Previous studies found that EB-induced reduction in adenogenesis was accompanied by alterations in growth factor networks that were associated with long-term alterations in uterine structure and function (see [16, 17]). Representative photomicrographs are presented in Figures 6 and 7, and effects of estrogen treatment on uterine mRNAs and protein are summarized in Table 2. As observed in EV-exposed ewes from study 1, EB exposure during both periods increased WNT2 mRNA expression on P28 and P56 (Fig. 6). However, the transient effects of EB to increase WNT2 mRNA were not permanent, because WNT2 mRNA was not different in the uteri of control and EB-exposed ewes on P112. WNT2B mRNA was ablated in the stroma by EB exposure on P28 and P56, but WNT2B mRNA was not different on P112 in uteri from CX and EB ewes. EB exposure from P14 to P28 did not affect WNT5A mRNA, but WNT5A was decreased in ewes receiving EB from P42 to P56. Interestingly, exposure of ewes to EB from P42 to P56 resulted in decreased WNT5A mRNA in the endometrial GE on P112. No transient effect of EB exposure was detected on WNT7A mRNA in the endometrial LE on P28 or P56; however, WNT7A mRNA was much reduced in the uterine LE on P112 in EB as compared to CX ewes.


Figure 6
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  		FIG. 6. Effects of EB exposure from P14 to P27 (period 1) or P42 to P55 (period 2) on uterine WNT gene expression. In each panel portion, representative photomicrographs of in situ hybridization results are presented in darkfield illumination. The black melanocytes in the uterus appear white under darkfield illumination. Car, Caruncle; GE, glandular epithelium; LE, luminal epithelium; S, stroma. Bar = 100 µm.


Figure 7
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  		FIG. 7. Effects of EB exposure from P14 to P27 (period 1) or P42 to P55 (period 2) on uterine SFRP2 mRNA. In each panel portion, representative photomicrographs of in situ hybridization results are presented in darkfield illumination. The black melanocytes (Mel) in the uterus appear white under darkfield illumination. Car, caruncular endometrium; GE, glandular epithelium; LE, luminal epithelium; S, stroma. Bar = 100 µm.

As observed in EV-exposed ewes in study 1, transient exposure of neonatal ewes to EB from P14 to P28 increased SFRP2 mRNA in the caruncular areas (Fig. 7). Strikingly, EB exposure also increased SFRP2 mRNA in the stroma between the tips of the developing GE and the inner circular layer of the myometrium on P28. In contrast, exposure of ewes to EB from P42 to P56 did not affect SFRP2 mRNA in the uterus on P56. Long-term effects of EB exposure on uterine SFRP2 mRNA were not observed on P112, regardless of exposure period.

EB exposure during either period did not affect expression of WNT4, WNT11, FZD2, FZD6, LRP6, SFRP1, CTNNB1, GSK3B, CHD1, MSX1, or MSX2 mRNAs on P28, P56, or P112, regardless of EB dose (data not shown). Similarly, CTNNB1, p-JNK, JUN, and p-JUN, as well as TCF7L2 protein, in the neonatal ovine uterus were not affected by EB treatment (data not shown).

DISCUSSION

The canonical WNT/CTNNB1/TCF signaling pathway is activated by a number of WNTs identified in the neonatal ovine uterus, including WNT2, WNT2B, WNT4, and WNT7A (see Fig. 8). In the present study, FZD receptors 2 and 6, the LRP6 coreceptor, and GSK3B were expressed in all uterine cell types. Cytoplasmic CTNNB1 levels are normally kept low through continuous proteasome-mediated degradation, which is controlled by a complex containing GSK3B. When cells receive WNT signals, the degradation pathway is inhibited, resulting in accumulation of CTNNB1 in the cytoplasm and nucleus. Nuclear CTNNB1 interacts with transcription factors, most notably TCF7/LEF1 members, to affect transcription. In the present studies, CTNNB1 and TCF7L2 proteins were observed in all uterine cell types, but were most abundant in the endometrial LE and GE. Therefore, the canonical WNT signaling pathway appears to be active in all intercaruncular endometrial cell types, and particularly in the epithelia of the developing neonatal ovine uterus. Selected WNT/CTNNB1/TCF target genes include: JUN (c-Jun) [30], LEF1/TCF7 [31], MET [32], and MSX2 [33, 34]. In the present study, JUN was expressed in the nuclei of most endometrial cells, but was most abundant in LE and GE. MSX2 was expressed only in the uterine epithelia in the present study. MET is also expressed only in uterine epithelia of the neonatal ovine uterus [16, 54]. CTNNB1 is a critical component of the cadherin cell adhesion complex, although it also has a well-established role as an essential mediator of the canonical WNT signal transduction pathway. CTNNB1 and CHD1 (E-cadherin) is the major cell adhesion system in epithelia, and cell-cell adhesion is important for epithelial morphogenesis [55]. As expected, CHD1 was expressed only in the endometrial epithelia. Collectively, results suggest that the canonical WNT signaling pathway has a biological role in uterine growth and endometrial gland morphogenesis in the neonatal ovine uterus (Fig. 8).


Figure 8
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  		FIG. 8. Proposed source and actions of the WNT signaling system in postnatal uterine development in the sheep. A) Schematic illustrating autocrine and paracrine actions of WNTs and SFRP2 in the neonatal ovine uterus. In the intercaruncular endometrium, WNTs expressed in the luminal epithelium or LE (WNTs 5A, 7A, and 11) may have autocrine or paracrine actions on the LE or stroma, respectively. WNT2B is expressed only in the stroma and may have autocrine or paracrine actions on the stroma or on the LE and GE, respectively. WNT5A is expressed predominantly by the GE and may have autocrine or paracrine actions on the GE or stroma, respectively. SFRP2 is produced predominantly by the stroma of the caruncles as well as by the intercaruncular stroma between the tips of the glands and the inner circular layer of the myometrium. Binding of the FZD receptors for the WNTs on the epithelia and stroma inhibit epithelial growth and development into the caruncular areas of the endometrium as well as the myometrium. The specific effects of each WNT and SFRP2 during ovine uterine morphogenesis depend on the temporal and spatial alterations in expression observed during postnatal development as summarized in Table 2. B) Schematic illustrating proposed model for the activation of the canonical and noncanonical WNT signaling pathways and inhibition of those pathways by SFRP2 in the endometrium of the neonatal ovine uterus. Based on the biological effects of WNTs in other organs and model systems, activation of the canonical signaling stimulates epithelial adhesion and proliferation as well as stromal cell proliferation in the intercaruncular endometrium. Activation of the noncanonical pathway would stimulate epithelial cell movement. Estrogen ablation and inhibition of endometrial adenogenesis was associated with an increase in stromal SFRP2 and a decrease in WNT2B, WNT7A, and WNT11 (see Table 2), which would decrease cell proliferation and movement by inhibiting canonical and noncanonical WNT signaling in the intercaruncular areas of the endometrium.

The canonical WNT signaling pathway is important for Müllerian duct differentiation and postnatal uterine development in mice [44, 46, 56]. In mice, Wnt7a is also exclusively expressed in the LE cells of the uterus after birth [43, 48]. Wnt7a-null mutants are viable and exhibit malformations in the female reproductive tract [43, 48, 57]. The Wnt7a-null uterus has a stratified (in contrast to a simple columnar) endometrial LE surrounded by a shallow stromal layer that does not contain glands [48]. The myometrium appears to be hyperplastic and disorganized by 3 mo of postnatal development, and by 6 mo, the endometrial stroma is displaced by the myometrium [48, 58]. Recently, Carta and Sassoon [59] provided evidence supporting the hypothesis that Wnt7a coordinates a variety of cell and developmental pathways that guide postnatal uterine growth and hormonal responses, and that disruption of these pathways by diethylstilbestrol (DES), an ESR1 agonist and endocrine disruptor, leads to aberrant cell death. In the present study 2, exposure of neonatal ewes to EV from birth completely ablated endometrial adenogenesis and decreased WNT7A mRNA in the LE and WNT2B in the stroma. In study 3, EB exposure during periods 1 (P14–P28) and 2 (P42–P56) reduced gland development. EB retardation of endometrial adenogenesis was associated with ablation of WNT2B mRNA in the stroma. Although WNT7A mRNA was not acutely affected by EB exposure, the long-term effects of EB exposure were to repress WNT7A mRNA in the LE. The uterine phenotypes of Wnt7a-null female mice resemble those of wild-type female mice that are prenatally treated with DES, a potent ESR1 agonist [58]. Subsequent studies have shown that perinatal downregulation of Wnt7a expression might account for the uterine defects that are observed in DES-treated females [58]. Thus, inappropriate exposure of mice and sheep to estrogens during critical developmental periods disrupts WNT7A gene expression in association with permanent alterations in uterine epithelial morphogenesis. In the neonatal sheep, estrogen exposure also disrupted normal patterns of WNT2B expression and prematurely induced WNT2, which is normally expressed only in the endometrial stroma of the adult ovine uterus (unpublished results). It is possible that the signaling pathways activated by WNT2 and WNT2B are not redundant and exhibit cross-talk with other WNTs. In study 1, EV inhibition of adenogenesis was associated with reductions of TCF7L2 protein in the uterine epithelia, which can be correlated to the loss of LEF1/TCF7 target genes, including TCF7L2 itself and MSX1 as well as MET [16]. Collectively, these studies suggest that the canonical WNT/CTNNB1/TCF7 signaling pathway is a conserved regulator of uterine development and postnatal endometrial gland morphogenesis.

In addition to the canonical pathway, the noncanonical or planar cell polarity pathway activated by WNT5A and WNT11 also has a biological role in uterine differentiation. In mice, Wnt5a is expressed in the mesenchyme of the uterus, cervix, and vagina [43]. In the present studies, WNT5A was found to be expressed in the LE and then predominantly in the GE of the neonatal ovine uterus, with low levels in the stroma. Wnt5a mutant mice have short and coiled uterine horns of normal diameter, lack defined cervical/vaginal structures, and die at birth because of a failure to complete anteroposterior body axis development [60]. To circumvent the neonatal lethality, the female reproductive tract from Wnt5a-null mice was grafted into adult hosts to assess postnatal potential and phenotypes [49]. Although the oviduct, uterus, and cervix developed in the absence of Wnt5a, the mutant uterus failed to form glands. WNT11 has not been reported in the mouse or human uterus.

WNT5A and WNT11 activate the noncanonical or planar cell polarity pathway [61–64] and antagonize the canonical WNT/ß-catenin pathway in Xenopus embryos and mammalian cells [61, 63, 64]. The noncanonical or planar cell polarity pathway mediates cell polarity and cell movement via modification of the actin cytoskeleton by activation of the small GTPases Rac and Rho [35–38]. Rac activation stimulates JNK activity [39–41] that plays essential roles in organogenesis by regulating cell survival, apoptosis, and proliferation [42]. Our studies in the neonatal uterus indicate that activated JNK and JUN are present in the developing endometrium. Cell polarity and movement are undoubtedly critical for endometrial development and adenogenesis [2, 65]. In study 2, estrogen ablation of endometrial gland differentiation was associated with a loss of WNT5A and WNT11 expression in the endometrial epithelium. In study 3, estrogen inhibition of endometrial adenogenesis was also associated with an exposure period-dependent loss of WNT5A in the endometrial GE. These results support the idea that the noncanonical signaling pathways activated by WNT5A and WNT11 in the neonatal ovine uterus govern endometrial development and adenogenesis in a period- or stage-dependent manner (Fig. 8).

The importance of WNT regulation of endometrial gland morphogenesis is strongly supported by the discovery that SFRP2, an inhibitor of WNT binding to FZD receptors, is expressed only in the stroma of the caruncular areas of the endometrium that are aglandular. In the ruminant uterus, endometrial glands are restricted spatially to developing in discrete areas of the endometrium that become the intercaruncular areas of the endometrium and do not develop into the inner circular layer of the myometrium [1, 2, 8]. In study 1, SFRP2 mRNA was almost undetectable in the endometrium and then was induced in the stroma of the aglandular caruncular areas of the endometrium by P7, which is the onset of budding differentiation of the GE in the intercaruncular areas of the endometrium. Further, SFRP2 mRNA was localized in an area between the tips of the glands and the inner circular layer of the myometrium on P28 to P56. These observations support the hypothesis that SFRP2 restricts WNT actions to the intercaruncular areas of the endometrium, thereby specifying the location of endometrial adenogenesis in the neonatal ovine uterus. This hypothesis is supported by studies 2 and 3. In study 1, EV ablation of endometrial gland differentiation was associated with an increase in SFRP2 mRNA in the stroma, particularly in the area underneath the LE. In study 2, estrogen inhibition of endometrial gland development was associated with an increase in SFRP2 mRNA in the caruncular stroma as well as in the stroma of the intercaruncular areas, particularly between the tips of the glands and the inner circular layer of the myometrium. The aberrant induction of SFRP2 was coincidental with estrogen-induced reductions in several WNTs. Given that SFRP2 inhibits WNT activation of both canonical and noncanonical signaling pathways, results of the present studies support the hypothesis that SFRP2 restricts WNT signaling and actions to the intercaruncular endometrium by inhibiting gland development from the LE in the caruncular endometrium and preventing GE development into the myometrium in the intercaruncular areas of the endometrium. Indeed, overexpression of Sfrp2 in the mouse uterus inhibited uterine epithelial cell growth, presumably by inhibiting activity of Wnt4 and Wnt5a [66].

Collectively, results of the present studies indicate that multiple WNTs are expressed in the neonatal ovine uterus and that both the canonical and the noncanonical WNT signaling pathways are candidate regulators of postnatal uterine development and endometrial adenogenesis. The spatial differences in WNT expression in the neonatal uterus suggest that they regulate endometrial differentiation via autocrine and paracrine effects on the epithelium and/or stroma (Fig. 8A). Epithelial-stromal interactions are crucial for the development of a number of epitheliomesenchymal organs, including those derived from the Müllerian duct [67, 68]. In the mouse uterus, the capacity of uterine mesenchyme to support or induce Msx1 expression in Müllerian epithelium is correlated with mesenchymal expression of Wnt5a [69]. Recently, Huang et al. [70] found that Msx2 is required for the proper expression of several genes in the uterine epithelium including Wnt7a. Future experiments will focus on the role of the WNT signaling pathways and other homeobox transcription factors, such as MSX1 and MSX2, in differentiation and development of the neonatal ovine uterus.

ACKNOWLEDGMENTS

Authors thank Mr. Kendrick LeBlanc and other members of the Spencer laboratory for assistance with animal husbandry and surgeries.

FOOTNOTES

1 Supported by NIH grants HD38274 and P30 ES09106. Back

2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; tspencer{at}tamu.edu Back

Received: 28 November 2005.

First decision: 19 December 2005.

Accepted: 29 December 2005.

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