| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Female Reproductive Tract |
Brookdale Department of Developmental, Cellular and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), Hox, and other WNT genes, reveals either abnormal spatial distribution of transcripts or abnormal regulation in response to DES exposure. Taken together, the results of the present study demonstrate that Wnt7a coordinates a variety of cell and developmental pathways that guide postnatal uterine growth and hormonal responses and that disruption of these pathways leads to aberrant cell death.
apoptosis, developmental biology, estradiol, female reproductive tract, Müllerian ducts
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The uterus expresses high levels of estrogen receptor (ER) and progesterone receptor (PR), which are required for adult function as well as for mediating the hormone-driven remodeling that accompanies the estrous cycle [11– 15]. Consistent with a role in the adult, we find that Wnt gene expression fluctuates during estrus in the uterus [6]. In particular, Wnt7a levels decline during peak levels of estradiol secretion, suggesting that endogenous estrogenic stimuli repress Wnt7a expression [6]. Perinatal exposure of wild-type female mice to the synthetic estrogen-agonist diethylstilbestrol (DES) leads to significant perturbations in the postnatal development of the uterus [10, 16–18]. By 1– 2 mo after birth, DES-exposed uteri display morphological characteristics almost identical to those found in Wnt7a mutant uteri, including stratified epithelium, lack of uterine glands, decreased uterine stroma, and an enlarged and disorganized myometrium [5, 10]. When DES-exposed uteri are examined at birth, we find that Wnt7a expression is repressed, which is consistent with a model whereby precocious exposure to estrogenic stimuli results in down-regulation of Wnt7a during a critical period of perinatal patterning [10]. However, several differences between the DES model and the Wnt7a mutant remained to be addressed. In particular, DES-exposed uteri are larger at birth compared to nonhormone-exposed controls, whereas the Wnt7a mutant uterus is smaller [10]. One explanation for these differences is that an estrogen-mediated signal mediates the down-regulation of Wnt7a, which underlies morphogenetic programs ,and concomitantly acts through a Wnt7a-independent pathway to mediate tissue growth. If this model is correct, we would anticipate that sexually mature mutant uteri would grow in response to endogenous estrogens, which is not the case. It is also possible that the change in epithelial cell type (columnar to stratified) underlies a more fundamental disruption of the epithelial-mesenchymal interactions that are required for normal postnatal growth and estrogen responsivity. Alternatively, it is possible that the absence of Wnt7a expression during a critical perinatal period of development leads to a "reprogramming" of the uterus such that subsequent growth stimuli, such as estrogen, fail to execute appropriate cellular responses.
To address this question, we chose 2-wk-old female mice corresponding to a stage of normal high rates of uterine growth before high levels of ovarian hormone secretion that occur by 6 wk after birth and correspond to a stage of high sensitivity to exogenous estrogenic stimulation [19]. This stage is also ideal for assessing the uterotrophic response to the potent estrogen-agonist DES, which normally elicits a rapid increase in uterine size and cell division. At this stage, the Wnt7a mutant uterus shows neither abnormal epithelium nor overtly disorganized myometrium, thus presenting a situation in which a response to DES can be measured and compared with wild-type uteri. In the Wnt7a mutant uterus, we do not see the expected increase in cellularity in response to DES. We find that the mutant uterus is still capable of undergoing extensive cell proliferation in response to DES; however, we detect variable levels of cell death in the mutant uteri, which become uniformly high in response to DES. Analyses of the expression patterns of a variety of patterning genes as well as ER in the mutant uterus closely resemble the pattern of uteri exposed to DES in utero. We propose that Wnt7a is required to mediate normal growth in the absence of estrogenic stimuli and is also required during the initial estrogen response, which results in an increased cellularity of uterine tissue.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All handling, housing, and care and feeding of mice used for the present study were carried out in accordance with National Institutes of Health (NIH) and institutional guidelines.
Chemicals
The DES was dissolved in a 10% ethanol saline solution (NaCl) at a concentration of 50 µg/ml (Sigma-Aldrich, St. Louis, MO).
Animals and Treatment
The 129 SV6/SvEv mouse strain was used for the experiments. All animals were housed in a temperature-controlled room (21–22°C) with a 12L:12D photoperiod. Mice were provided fresh reverse-osmosis/deionized water and NIH-31 lab chow (NIH, Bethesda, MD) ad libitum. Immature female mice (age, 15 days) were injected with DES (50 µg/ml in 10% ethanol saline solution; 
600 ng/g body wt). Control mice were injected with 10% ethanol saline solution alone. For early-phase analysis, mice were given a single injection and killed after 15 h. For late-phase analysis, mice were injected daily for three consecutive days and killed 72 h after the first injection. Mice were killed by cervical dislocation. All animal procedures were performed in accordance with guidelines outlined by the NIH.
Tissue Processing and Cell Counts
Reproductive tracts were dissected in PBS and blotted to remove excess buffer surrounding the tissue. Wet weight was determined with a precision balance. We note that wild-type and mutant uteri did not show any loss of fluids in the lumen, because our subsequent histological analyses did not show any sign of collapse of the overall diameter of the uterus. Thus, any increase in weight solely caused by retention of fluids would have been accurately measured. Tissues were fixed overnight in 4% PBS-buffered paraformaldehyde. After dehydration, tissues were embedded in paraffin, sectioned (thickness, 7 µm), and stained with hematoxylin and eosin. Comparative photographs of wild-type and mutant saline- or DES-treated mice were taken at the same magnification. Total cell numbers were counted per single compartment in whole-uteri cross sections. Three sections per individual and three individuals per group were counted. Statistical evaluation was made using the unpaired t-test [20, 21]
For 5-bromodeoxyuridine (BrdU)- and TUNEL-labeled cells, positive versus total cell ratios were calculated per tissue compartment in whole-uteri cross sections. Three sections per individual and three individuals per group were counted. Statistical evaluation was made using the unpaired t-test [20, 21].
In Situ Hybridization
In situ hybridization was performed as described previously [22]. Antisense 35S-labeled riboprobes were generated for Wnt7a and Wnt4 [23], Wnt5a [24], smooth muscle heavy chain [25], HoxA.10 [26], HoxA.11 [27], ER [28], and PR [29].
Immunohistochemistry
Mouse female reproductive tracts were fixed in 4% PBS-buffered paraformaldehyde overnight. After dehydration, specimens were embedded in paraffin and sectioned (thickness, 7 µm). Sections were blocked for 1 h in 10% goat serum, 0.1% Triton X-100 1% BSA, and 0.2% gelatin and then incubated for 1 h with a monoclonal anti-BrdU (1:50 in blocking buffer; Roche Applied Science, Indianapolis, IN). Sections were washed and incubated in biotin-conjugated anti-mouse immunoglobulin G secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 1:500 dilution in blocking buffer for 1 h (room temperature). Bound mouse antibodies were detected using Streptavidin-HRP (Zymed Laboratories, San Francisco, CA) with aminoethyl carbazole as substrate. Sections were mounted on glass slides with coverslips (GVA-mount; Zymed Laboratories) before imaging.
Cell Death Assays
TUNEL assay for DNA fragmentation was done using the In Situ Cell Death ApopTag Detection kit (Intergen, Purchase, NY) according to manufacturer's protocol.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Two-week-old, postnatal wild-type and Wnt7a mutant uteri were dissected free of surrounding tissues, measured for wet weight, and underwent tissue fixation and standard paraffin histology. As shown in Figure 1, a cross section through the midrostral-caudal portion of a uterine horn revealed a clear difference in the diameter and overall size of the wild-type compared to the mutant uterus. At this stage, the only major histological difference in appearance, other than diameter, was the complete lack of uterine glands in the mutant as previously described [5, 10] (Fig. 1). This difference in size was not reflected in the length of the uterine horns, which was the same in both wild-type and Wnt7a mutant uteri (data not shown). Consistent with the difference in uterine diameter, we observed a twofold difference in the wet weights of the wild-type compared to the Wnt7a mutant uteri (Fig. 1). Counts performed directly from tissue sections showed a twofold (epithelial compartment) to almost fourfold (stromal and myometrial compartment) increase in the number of cells in the wild-type compared to the mutant uteri (Table 1). Thus, by 2 wk after birth, the wild-type uterus was larger in terms of size, weight, and cell number.
|
|
Wnt7a Mutant Uterus Has Decreased Levels of Cell Proliferation
We also addressed whether the greater cellularity of the wild-type compared to the mutant uterus reflects differences in basal levels of cell proliferation during postnatal growth. Two-week-old mice were subjected to a single injection of BrdU, killed 2 h later, and scored for labeled cells as previously described [30]. We note that these mice were injected with saline 15 h before the BrdU injection to serve as controls for hormone experiments described later. Labeled cells were counted in each cell type compartment and scored (Fig. 2 and Table 2). As seen in Figure 2, the epithelium showed a low baseline level of proliferation, and counts of this compartment showed no significant difference in labeling indices (Table 2). In contrast, the stroma and myometrium showed ninefold and threefold higher levels of proliferation, respectively, in the wild-type compared to the mutant uterus (Fig. 2 and Table 2). We also note that the uterine glands showed a high rate of proliferation in the wild-type uterus at this stage, which is consistent with the fact that glandular genesis occurs during postnatal development [31–33] (Fig. 2).
|
|
Wnt7a Mutants Display a Highly Abrogated Uterotrophic Response
The uterus is highly responsive to exogenous estrogenic stimuli, and it undergoes a marked increase in size and weight following even brief estrogen exposure [19, 34–38]. Within the first 15 h after estrogen exposure, a burst of cell proliferation, primarily in the epithelium, occurs and is followed by accumulation of water within the stroma and enlargement of the uterine glands by 72 h after either a single or repeated exposure [19, 30, 36, 38, 39]. We therefore exposed 2-wk-old wild-type, heterozygote, and Wnt7a mutant females to three daily exposures of DES and compared them with control (saline-injected) mice 24 h after the final injection (72-h time point). At 2 wk of postnatal development, wild-type uteri showed overt signs of cytodifferentiation, including glands as well as muscle and epithelial cells typical of the mature adult, even though circulating levels of steroid hormones were low [19] (Fig. 1). Furthermore, with the exception of the lack of uterine glands, the cytoarchitecture of the mutant uterus had an overall wild-type appearance in that neither the myometrium nor the epithelial compartments showed overt signs of disrupted morphology, which normally appear by 1–2 mo after birth (Fig. 1). Thus, any differences in responses to DES between the wild-type and mutant uterus likely were not caused by a difference in the cell types present in the two situations. The wild-type and Wnt7a heterozygote uteri displayed a marked increase in size and a fourfold to fivefold increase in wet weight following DES exposure compared to control (saline-injected) wild-type and Wnt7a heterozygote uteri (Fig. 3) (P < 0.05). In contrast, Wnt7a mutant uteri showed no increase in wet weight (Fig. 3). In wild-type (Fig. 3) and heterozygote uteri (data not shown), histological analyses revealed a thickening of the uterine wall following DES exposure. Thickening reflected both edema (i.e., accumulation of water) and an apparent increase in cellularity following estrogenic stimulation. In addition, uterine glands became hypertrophic and secretory following DES treatment (Fig. 3). In contrast, we observed that DES-exposed mutant uteri displayed an abnormal response consisting of enlargement of the uterine lumen accompanied by thinning of the uterine wall (Fig. 3). Taken together, our observations reveal that the uterotrophic response is markedly reduced and abnormal in the Wnt7a mutant.
|
Wnt7a Governs Cellularity and Cell Death in Response to DES
At the end of the uterotrophic assay (72 h), Wnt7a mutant uteri showed an increase in luminal size but no increase in weight or thickness of the uterine wall. Typically, a significant increase in cellularity was observed by 72 h in wild-type uteri that appeared to be absent in the mutant uteri. Thus, we quantified the cellularity per tissue compartment (epithelium, stroma, and myometrium) in uterine cross sections for all experimental groups at the 72 h, because changes in cellularity were pronounced at this time point (Table 1). Exposure to DES for 3 days led to a fivefold increase in cellularity in the wild-type epithelium compared to controls (P < 0.05), whereas the mutant uteri showed no significant change (Table 1). We did not see a significant increase in stromal or myometrial cellularity when comparing DES to control wild-type or mutant uteri (Table 1), which is consistent with previous observations that the epithelial cells of the lumen display the most robust proliferative response [39].
To evaluate the proliferative response, we used BrdU administered 15 h following a single injection of DES or saline, and we analyzed labeled cells 2 h after BrdU injection as previously described [39]. Stained sections from either control (Fig. 2) or DES-exposed wild-type uterus revealed numerous BrdU-labeled cells in all cell compartments (epithelium, stroma, and myometrium) (Fig. 4). Levels of proliferation were low in the control mutant uteri (Fig. 2), but we detected numerous labeled cells in DES-exposed mutant uteri (Fig. 4). These results indicate that the wild-type uterus maintains a higher baseline level of proliferation compared to the mutant uterus in the absence of hormonal challenge; however, the mutant uterine epithelium is capable of a proliferative response to DES. We also noted that the cellular compartments displayed different levels of proliferation in response to DES; thus, we quantified labeled cells in each compartment (Table 2). These data reveal that the wild-type uterus undergoes a significant increase in proliferation in the epithelial (13-fold), stroma (twofold), and myometrial (twofold) compartments (P < 0.05) (Table 2). In the DES-exposed mutant uteri, we observed a significant increase in labeled cells in the epithelial (20-fold), stromal (12-fold), and myometrial (fivefold) compartments (P < 0.05) (Table 2). Thus, we conclude that the lack of increased cellularity in the mutant uterus does not reflect a failure to proliferate in response to DES. Indeed, the mutant uterus shows somewhat higher levels of DES-induced proliferation compared to wild-type uterus.
|
Wnt7a Mutant Uterus Displays Elevated Levels of Cell Death
These data reveal that mutant uteri do not show an obvious defect in cell proliferation. Therefore, we investigated cell death using the in situ TUNEL assay followed by counts of labeled cells at the 72-h time point. First, we compared the wild-type and Wnt7a mutant uteri exposed to saline (Table 3). These counts revealed that levels of cell death in the wild-type uterus were uniformly low. In contrast, we found that levels of cell death in mutant uteri were highly variable (Table 3); thus, although a trend was observed toward higher baseline levels of cell death in mutant uteri, these differences were not statistically significant. We note, however, that we did not see a single wild-type uterus that displayed high levels of cell death. When wild-type uteri were exposed to DES, we did not see significant levels of cell death in any individual examined (Fig. 5 and Table 3). In sharp contrast, we observed a significant increase in the number of apoptotic cells in the epithelium (twofold, P = 0.05) and stroma (twofold, P = 0.08) of the mutant uteri following DES exposure as compared to DES-exposed wild-type uteri (Fig. 5 and Table 3). No significant change was detected in the myometrium (Table 3). Because our quantitative data revealed a uniformly high level of cell death in DES-exposed mutant uteri, whereas levels in the saline-exposed mutants displayed highly variable levels of cell death, we conclude that the mutant uterus responds to DES by triggering and/or maintaining high levels of cell death.
|
|
DES-Mediated Down-Regulation of Wnt7a Occurs after the Cell Proliferative Response
If Wnt7a plays a key role in the DES response, we predict that Wnt7a expression must be present during some phase of the DES response before its down-regulation [10]. Indeed, to our knowledge, the kinetics of the Wnt7a response have not been described previously. We therefore assessed Wnt7a transcript levels in the postnatal uterus at 15 h, during which time cell proliferation occurs, and at 72 h, the time point at which cell proliferation has ceased and the uterotrophic response is completed. The nonfunctional, recombined transcript can be followed in these assays of mutant uteri as previously reported [40]. We observed high levels of Wnt7a transcripts in both the mutant and wild-type uteri at 15 h following DES exposure (Fig. 6). In contrast, both mutant and wild-type uteri showed a decrease in Wnt7a transcripts 72 h after DES exposure (Fig. 6). These data reveal that the regulatory regions controlling transcription of Wnt7a in response to estrogens are intact in the mutant, and they further suggest that Wnt7a function is not required for DES-mediated down-regulation of the Wnt7a gene. These data also reveal that Wnt7a expression (i.e., transcription) is present during the first 15 h of the uterotrophic response.
|
Wnt7a Is Required for Proper Regulation of Other Patterning Genes in the Uterus
Diethylstilbestrol primarily acts via the ER isoform in the uterus, as demonstrated in ER knockout mice, which do not exhibit a uterotrophic response [41]. Therefore, we tested the simple hypothesis that the abrogated uterotrophic response in the Wnt7a mutant is caused by disruption of ER expression. In wild-type uteri, ER expression was primarily restricted to the uterine stroma, whereas we detected ER expression in all compartments of the mutant uterus (Fig. 7). By 72 h, DES exposure elicited a change in the distribution of the ER transcript in the wild-type and mutant uteri, with the highest levels detected in the epithelium and myometrium (Fig. 7). These data reveal that Wnt7a is required for the wild-type distribution of ER; however, mutant uteri expressed high levels of receptor in all tissue compartments (epithelium, stroma, and myometrium) and, thus, presumably are capable of responding to estrogenic hormones.
|
We have reported previously that Wnt4 and Wnt5a are expressed in the adult uterus, and like those of Wnt7a, their levels fluctuate or their patterns change during estrus [6]. We first established the baseline pattern of expression of all patterning genes examined for the present study at 2 wk postnatal; to our knowledge, this has not been previously described. In control wild-type mice, we observed a situation similar to that previously described for the wild-type adult uterus, in which Wnt4 expression was restricted to the stroma (Fig. 7) [6]. By 72 h after DES exposure, Wnt4 expression became restricted to the epithelium and myometrium (Fig. 7). In contrast, we detected Wnt4 expression in both the stroma and the epithelium in the mutant uterus, and this expression became restricted to the epithelium and myometrium following DES exposure (Fig. 7). Thus, the initial pattern of Wnt4 expression in the mutant uterus was abnormal, whereas DES exposure induced a pattern of Wnt4 expression similar to that of the DES-exposed wild-type uterus.
Examination of Wnt5a distribution at 2 wk postnatal in wild-type control uteri revealed Wnt5a expression localized primarily to the uterine stroma and at lower levels in the uterine epithelium (Fig. 7), similar to the situation in the adult [6]. Following DES exposure, we detected Wnt5a expression in all compartments (Fig. 7). Interestingly, we observed that the distribution of Wnt5a in the mutant was similar to the pattern observed in the wild-type, DES-treated uterus (Fig. 7), and no additional changes were observed following DES exposure (Fig. 7).
Studies have revealed that the homeogenes HoxA.10 and HoxA.11 are downregulated in response to DES, suggesting HOX gene involvement in DES-mediated malformations and estrogen responses in the normal uterus [14, 42, 43]. As reported previously by others [43], we observed in the present study that HoxA.10 was detectable at high levels in the uterine stroma and myometrium, and we further confirmed the observation that HoxA.10 levels declined in the stroma following DES exposure (Fig. 7). In contrast, DES did not downregulate HoxA.10 in the stroma of the mutant uterus (Fig. 7). Results identical to those obtained for HoxA.10 were observed in our in situ analysis for HoxA.11 (data not shown). These data indicate that DES-mediated downregulation of HoxA.10 and HoxA.11 expression in the uterus are downstream and dependent on Wnt7a.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mouse mutants to all three members of the WNT family that are expressed in the uterus have been generated. The phenotype of the Wnt4 mutant in the female reproductive tract has not been examined, because early urogenital development is severely disrupted [44]. The Wnt5a mutant mouse shows severe developmental phenotypes throughout the body and dies at birth [45]. These mice are currently under investigation in our laboratory, because they develop müllerian ducts. Wnt7a mutant mice are viable, reflecting the more discreet and tissue-specific restricted expression and requirements of this gene [40]. Because the mice are viable, we could readily assess the contribution of Wnt7a to uterine development.
Wnt7a expression is restricted to the luminal epithelium of the uterus throughout development, and levels are down-regulated in response to endogenous estrogen secretion as well as exogenous estrogenic stimulation, such as DES exposure [5, 10] (present study). We had determined previously that Wnt7a is down-regulated by DES and that down-regulation of Wnt7a is a primary mechanism by which DES induces malformations of the female reproductive tract following perinatal DES exposure [10, 46]. The observation that perinatal exposure to DES leads to a largely overlapping uterine phenotype with the Wnt7a mutant led to the discovery that Wnt7a is a key target in DES-mediated signaling events in the reproductive tract. However, these previous studies left critical issues unaddressed. In particular, DES exposure of wild-type female fetuses gives rise to uteri that are bigger than those of unexposed wild-type and Wnt7a mutants [10, 46]. One interpretation of these observations is that Wnt7a expression maintains a permissive environment by which DES can induce uterine growth. This model may be extended to include postnatal development, in which Wnt7a is necessary for the postnatal growth of the uterus that precedes ovarian hormone secretion.
The nature of a Wnt7a-mediated, permissive uterine environment likely is complex; however, we show in the present study that the mutant uterus displays a spatial pattern of gene expression for Wnt4 and Wnt5a that resembles the wild-type uterus, which has already been exposed to DES. In this manner, the spatial distribution of signaling factors is already perturbed in the mutant and may not be appropriately "set" to execute postnatal and DES-mediated growth. In support of this model, we find that HoxA.10 and HoxA.11 do not show the typical down-regulation in the mutant in response to DES exposure. This failure to down-regulate HoxA.10 and HoxA.11 is particularly striking, because these genes are no longer expressed in fully mature Wnt7a mutant uteri [5]. Thus, whereas Wnt7a is required for maintaining Hox gene expression in the adult uterus, it is also required, paradoxically, for Hox gene down-regulation in response to an estrogenic stimulus. We note that the HoxA.11 and Wnt7a mutant uteri share many phenotypic similarities, including the presence of stratified epithelium and the lack of glands in the adult [47, 48]. Taken together with observations from the present study, we propose that Wnt7a and HoxA.11 cooperate in a regulatory loop that guides morphogenetic processes during postnatal development of the uterus. The down-regulation of HoxA.10 and HoxA.11 may be a critical step in the early maturation of the uterus, when it is first exposed to ovarian-secreted estrogens. The failure to down-regulate Hox genes appropriately may ultimately lead to the dysmorphogenesis that we have observed in the adult Wnt7a mutant uterus.
Our data reveal that ER transcripts are present in the mutant uterus in all tissue compartments, ruling out a simple explanation that Wnt7a is required for ER gene expression. It has been demonstrated previously that expression of the ER in uterine stroma is required for an estrogen response in the stroma as well as in adjacent epithelial cells [49]. Thus, whereas the precise spatial pattern of ER expression differs in the mutant uterus, ER gene expression is present in the mutant stroma. Therefore, the compromised uterotrophic response is not the result of a fundamental perturbation in ER-mediated signaling.
It has been demonstrated that Wnt7a down-regulation by DES requires a functional ER [14], placing ER upstream of Wnt7a regulation. In the present study, we demonstrate that Wnt7a transcripts remain at high levels in the wild-type and mutant epithelium during the first 15 h of the uterotrophic response; however, by 72 h, Wnt7a transcripts are no longer detected. Thus, Wnt7a is present during cell proliferation, even though it does not appear to be required, because Wnt7a mutant uteri show robust proliferation in response to DES. The down-regulation of Wnt7a by 72 h may reflect a negative-feedback regulatory mechanism that ultimately terminates the response.
Previous reports have shown that WNT-signaling effectors control cell proliferation and confer resistance to cell death in pre-B lymphocytes [50]. Furthermore, Wnt1 prevents apoptosis induced by chemotherapeutic drugs [51], and down-regulation of Wnt3a leads to an increased rate of cell death in the mouse embryo tail bud [52]. The present study reveals that Wnt7a acts as a suppressor of cell death mediated by DES and, possibly, other endogenous signals underlying postnatal growth, including low levels of circulating steroids. Whereas cell proliferation underlies the increase in cellularity following estradiol exposure, cell death underlies the decrease in cellularity that returns the uterus to its initial state by the end of metestrus [53–57]. In the absence of Wnt7a, DES is still able to induce proliferation in the immature uterus; however, this response is coincident with an aberrant and pronounced cell death response. Low levels of proliferation (data not shown) and high levels of cell death are seen 72 h after DES exposure in mutant uteri, suggesting that cell proliferation precedes cell death in the uterotrophic response. This explains the observation that the Wnt7a mutant uterus does not show a net increase in cellularity following DES exposure and may extend to normal postnatal development. Thus, it is possible that Wnt7a participates in coordination of the multiple and complex cellular responses that underlie uterine responses to circulating ovarian hormones. Indeed, in the absence of Wnt7a, we note that the levels and spatial distribution of the expression of other regulatory genes are disturbed and more closely resemble those of uterine tissue that has been exposed to an estrogenic stimulus.
We suggest that in the absence of a coordinated response to ovarian hormones, cellular conditions arise that could promote tumor formation. Consistent with this notion, neonatal DES exposure in hamsters and mice followed by estradiol stimulation promotes apoptosis and endometrial adenocarcinomas [58–61]. Our data are entirely consistent with these observations in that neonatal exposure to DES replicates many aspects of the Wnt7a mutant phenotype in the uterus. Thus, a perinatal disruption of Wnt7a expression caused by endocrine exposure "reprograms" the uterus to aberrantly respond to hormonal stimulation that occurs later in postnatal life. Taken together, our observations point to a tightly coordinated regulation of patterning genes in the uterus that guide development and establish the appropriate responses to hormonal cues that govern adult function. We further note that the cell death seen in the Wnt7a mutants in response to estrogenic stimulation, coupled with cell proliferation, provides a context whereby tumorigenic cells can be generated. These data suggest that Wnt7a acts as a tumor suppressor in the female reproductive tract. We are currently pursuing studies to explore this possibility further.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: David Sassoon, Brookdale Department of Developmental, Cellular and Molecular Biology, Mount Sinai School of Medicine, 1 G. Levy Place, New York, NY 10029. FAX: 212 860 9279; david.sassoon{at}mssm.edu
Received: 15 December 2003.
First decision: 8 January 2004.
Accepted: 18 March 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ERß, in estrogen target tissues in vivo through the use of an ER-selective ligand. Endocrinology 2002 143:4172-4177 and ß isoforms in rabbit uterus and ovary. J Cell Biochem 2001; 82:467-479[CrossRef][Medline] knockout mice exhibit resistance to the developmental effects of neonatal diethylstilbestrol exposure on the female reproductive tract. Dev Biol 2001 238:224-238[CrossRef][Medline] knockout mouse uterus. Proc Natl Acad Sci U S A 1999 96:3646-3651This article has been cited by other articles:
|
|
 |
|
  X. Hong, L. J. Luense, L. K. McGinnis, W. B. Nothnick, and L. K. Christenson Dicer1 Is Essential for Female Fertility and Normal Development of the Female Reproductive System Endocrinology, December 1, 2008; 149(12): 6207 - 6212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-W. Jeong, K. Y. Lee, S. J. Han, B. J. Aronow, J. P. Lydon, B. W. O'Malley, and F. J. DeMayo The p160 Steroid Receptor Coactivator 2, SRC-2, Regulates Murine Endometrial Function and Regulates Progesterone-Independent and -Dependent Gene Expression Endocrinology, September 1, 2007; 148(9): 4238 - 4250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Katayama, K. Ashizawa, T. Fukuhara, M. Hiroyasu, Y. Tsuzuki, H. Tatemoto, T. Nakada, and K. Nagai Differential Expression Patterns of Wnt and {beta}-Catenin/TCF Target Genes in the Uterus of Immature Female Rats Exposed to 17{alpha}-Ethynyl Estradiol Toxicol. Sci., June 1, 2006; 91(2): 419 - 430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hayashi and T. E. Spencer WNT Pathways in the Neonatal Ovine Uterus: Potential Specification of Endometrial Gland Morphogenesis by SFRP2 Biol Reprod, April 1, 2006; 74(4): 721 - 733. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J Kimber Leukaemia inhibitory factor in implantation and uterine biology Reproduction, August 1, 2005; 130(2): 131 - 145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Ruden, L. Xiao, M. D. Garfinkel, and X. Lu Hsp90 and environmental impacts on epigenetic states: a model for the trans-generational effects of diethylstibesterol on uterine development and cancer Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R149 - R155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-W. Huang, Y. Yin, Q. Bi, T.-C. Chiang, N. Garner, J. Vuoristo, J. A. McLachlan, and L. Ma Developmental Diethylstilbestrol Exposure Alters Genetic Pathways of Uterine Cytodifferentiation Mol. Endocrinol., March 1, 2005; 19(3): 669 - 682. [Abstract] [Full Text] [PDF]
|
|
| ||||
