Emx2 Regulates Mammalian Reproduction by Altering Endometrial Cell Proliferation

Hugh S. Taylor and Xiaolan Fei

Yale University School of Medicine, New Haven, Connecticut 06520-8063

Address all correspondence and requests for reprints to: Hugh S. Taylor, Division of Reproductive Endocrinology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, Connecticut 06520-8063. E-mail: hugh.taylor{at}yale.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The molecular mechanisms that underlie embryo implantation are poorly understood. Under the control of sex steroids, uterine endometrium undergoes tremendous, yet tightly controlled, proliferation in each estrous cycle to facilitate implantation; disorders of endometrial proliferation underlie several uterine diseases. We have previously identified the Emx2 gene as a transcriptional target of HOXA10 regulation in the reproductive tract. Here we report the function of Emx2 in murine implantation and regulation of endometrial proliferation. We transfected mice on d 2 post coitus with pcDNA3.1/Emx2, Emx2 antisense, or respective controls consisting of empty pcDNA3.1 or a random order oligonucleotide by intrauterine lipofection. Increased expression of Emx2 reduced average implantation rate by approximately 40% (P = 0.00006) resulting in an average number of implanted embryos per litter of 13.7 in the control group to 8.2 in the pcDNA3.1/Emx2-treated group. Neither treatment altered the number of mice attaining pregnancy with at least one embryo. Decreased Emx2 expression did not alter litter size. Neither treatment affected the birth weight of the pups. To elucidate potential mechanisms through which Emx2-regulated reproduction, markers of endometrial differentiation, proliferation, and apoptosis were assessed. Increased Emx2 expression significantly decreased endometrial cell proliferating cell nuclear antigen expression and 5'-bromo-2' deoxyuridine incorporation. Markers of stromal cell differentiation (IGF binding protein-1, prolactin), epithelial differentiation (calcitonin), and apoptosis (activated caspase3) were unchanged. In human endometrial epithelial cells in vitro, Emx2 reduced cell number indicating diminished proliferation. Emx2 controls mammalian reproduction by adjusting endometrial cell proliferation without effecting differentiation. Regulated uterine Emx2 expression is necessary during reproduction for maximal implantation and litter size.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EMX2 IS THE ORTHOLOG of the Drosophila empty spiracles (ems) homeobox gene. Empty spiracles regulates Drosophila head development and development of the filzkörper in the posterior of the embryo (1, 2, 3, 4). The vertebrate Emx2 gene is located outside of the HOX cluster and its role in vertebrate central nervous system development is well characterized (5). Emx2 is essential for development of the dorsal telencephalon; targeted disruption leads to absence of the dentate gyrus and telencephalon, and greatly reduced size of the hippocampus and medial limbic cortex (6, 7). As with its Drosophila ortholog, Emx2 has a bimodal expression pattern; Emx2 is expressed in the developing vertebrate brain as well as the urogenital system (8, 9). Specifically, it is expressed in the epithelial components of the pronephros, mesonephros, ureteric buds, and the Wolffian and Müllerian ducts. Emx2 is also expressed in high levels in the gonads. Emx2 homozygous mutant mice die soon after birth due to absence of renal function. The role of Emx2 in reproductive tract development and function is still poorly characterized.

Emx2 expression persists in the adult urogenital tract. Specifically, Emx2 is expressed in the uterine endometrium in an estrous cycle phase-specific manner (10). In the human uterus, EMX2 expression is down-regulated at the time of endometrial remodeling and blastocyst implantation. Similarly, EMX2 expression is more abundant in the endometrium of postmenopausal women and decreased in endometrial tumors, correlating inversely with the extent of proliferation (11, 12). In endometriosis, a condition characterized by abnormal growth of endometrium, EMX2 expression is also decreased in the endometrium (13). We hypothesized that Emx2 has an essential function in vertebrate reproduction and in the regulation of endometrial cell proliferation or differentiation. Studies of genetic regulation of reproduction by this gene have been indirect because mice deficient for Emx2 die perinatally before the emergence of reproductive function. Therefore, to investigate the role of Emx2 in adult reproductive function, we altered the expression of the Emx2 gene in the adult murine endometrium.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Altered Emx2 Expression Affects Murine Litter Size
We have previously shown that endogenous Hoxa10 expression in the female reproductive tract leads to decreased Emx2 expression during the time of embryo implantation (10). To assess whether naturally occurring down-regulation of Emx2 expression during embryo implantation is essential to this process, a construct was designed that constitutively expressed Emx2 in the uterine endometrium. The uteri of 25 mice were transfected with this construct after detection of a vaginal plug. Twenty-five control mice were transfected with empty pcDNA3.1 plasmid. To ensure that this treatment had no unintended effect on the untreated ovaries or on oocyte production, embryos were flushed from the oviducts of five treated and five control animals; an equivalent number of embryos were obtained in each group, indicating that differences in litter size reflect implantation rate. Increased Emx2 expression in the uterus was documented by Northern analysis quantitative real-time RT-PCR. Emx2 expression (normalized to actin) was increased by 30- to 50-fold over endogenous levels (13, 14). Twenty mice in each treatment group proceeded to attempted pregnancy; an equal number of mice conceived in each group. As is shown in Fig. 1Go, increased expression of Emx2 resulted in a decrease in litter size (P = 0.00006, t test). Constitutive Emx2 expression resulted in a 40% decrease in litter size; the average number of implanted embryos at d 9 was decreased from 13.7 in the control group to 8.2 in the Emx2-treated group. The litter size ranged from 10 to 17 in the control group and from zero to nine in the Emx2-treated group. The total number of pregnant mice did not vary significantly between control and Emx2-treated mice. The weight of the pups also did not vary between groups. Histology revealed no change in uterine architecture or cytology between groups. The naturally occurring down-regulation of Emx2 in the reproductive cycle is essential for normal embryo implantation.



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Fig. 1. Emx2 Expression Diminishes Litter Size

A, Mice transfected with either an Emx2 expression construct (Emx2) or empty vector (Ctl) display similar pregnancy rates. Error bars are SEM. B, Litter size was reduced by 40% in mice transfected with Emx2 (n = 20 per group). *, Significant difference from control; P = 0.00006. C, The birth weight of pups born after Emx2 or control treatment did not differ. n = 130 and 80 in the Emx2 and control groups, respectively. D, Endometrial histology did not vary between groups. Normal glandular and stromal histologic development in the Emx2 transfected murine endometrium is demonstrated. Inset shows representative control. Scale bar, 75 µm.

 
To evaluate the effect of diminution of Emx2 expression in the adult murine uterus, we transfected the uterus with a phosphothiorate-modified antisense oligonucleotide complementary to the transcription start site of Emx2. Thirty mice were treated with Emx2 antisense, and thirty control mice were treated with a control oligonucleotide of the same length and nucleotide composition, in scrambled order, however. As described above, each uterine horn was transfected with the antisense or missense DNA/liposome construct 1 d after vaginal plug detection. Antisense Emx2-treated mice had Emx2 mRNA levels of approximately half that of missense-treated animals. The number of pregnant mice as well as litter size were measured at d 9 of pregnancy. Birth weights were obtained within 12 h postpartum. As shown in Fig. 2Go, down-regulation of Emx2 expression with antisense treatment did not alter litter size. Additionally, the number of mice pregnant is did not vary between the groups. The uterine histology was not affected by the transfection. There were no differences in weight or development between pups transfected with the Emx2 antisense, missense, or untreated animals.



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Fig. 2. Emx2 Antisense Does Not Alter Pregnancy

A, Pregnancy rate was similar in mice transfected with Emx2 antisense (AS) or missense control (Ctl) (n = 30 mice per group). B, Litter size at d 9 of gestation was similarly not effected by antisense treatment. C, The birth weight of pups born after antisense or control treatment did not differ (n = 120 and 145 in the missense and antisense groups, respectively). D, Uterine endometrial histology was not altered by treatment. Normal glandular and stromal development was observed in each group. Scale bar, 100 µm.

 
Increased Emx2 Expression Does Not Alter Markers of Endometrial Cell Differentiation
To elucidate potential mechanisms by which increased Emx2 might control endometrial function, and receptivity to the implanting blastocyst, we augmented Emx2 expression and assessed expression of markers previously implicated in endometrial differentiation and function. Immunohistochemistry (IHC) was performed to evaluate markers of endometrial cell differentiation and reproductive competence. Animals treated as described above were killed on d 3 after vaginal plug detection. The uteri were removed, fixed, and sectioned for IHC. Glandular cells express calcitonin in the receptive phase. Stromal cells express IGF binding protein-1 (IGFBP1) and prolactin in the receptive phase of the reproductive cycle. Immunohistochemistry was performed for each of these three markers. As is shown in Fig. 3Go, there were no differences in endometrial glandular cell calcitonin expression or endometrial stromal cell IGFBP1. There were also no differences in prolactin expression (data not shown). Untreated wild type mice, empty pcDNA 3.1 transfected controls, and pcDNA 3.1 Emx2 transfected mice all expressed these markers of differentiation at high level. Constitutive expression of Emx2 did not alter these well-characterized markers of endometrial differentiation.



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Fig. 3. Emx2 Expression Does Not Alter Endometrial Glandular or Stromal Differentiation in the Murine Endometrium

IHC identified markers of differentiated receptive endometrium after transfection with the Emx2 expression vector (Emx2) or empty control vector. Calcitonin, a well-characterized marker of endometrial glandular epithelial differentiation was unchanged by altered Emx2 expression. Similarly, IGFBP1, a marker of stromal cell differentiation and decidualization, was not altered by constitutive Emx2 expression. Scale bar, 100 µm.

 
Effect of Emx2 on Endometrial Cell Proliferation and Apoptosis
To evaluate the effect of Emx2 on endometrial cell proliferation and apoptosis, well-characterized markers of each of these processes were evaluated. Proliferating cell nuclear antigen (PCNA) was used to evaluate proliferation, whereas activated Caspase3 was used to evaluate apoptosis by immunohistochemistry. Mice were treated as described above. In addition, three mice were administered 5'-bromo-2' deoxyuridine (BrdU) 60 min before they were killed.

Increased Emx2 expression decreased cell proliferation as measured by PCNA immunochemistry, as shown in Fig. 4Go. A 5% decrease in this marker was seen in endometrial stromal cells. A 95% decrease in PCNA-positive cells was seen in glandular epithelial cells. Similar results were obtained using BrdU; there was a dramatic decrease in BrdU uptake in epithelial cells and a modest, but significant, decrease in the stromal cells. Total uterine BrdU uptake was reduced by approximately 50% (P < 0.001). EMX2 decreases uterine epithelial cell proliferation.



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Fig. 4. Emx2 Regulates Endometrial Proliferation

A, PCNA was highly expressed in the glandular cells after transfection with the empty pcDNA3.1 vector (Control). Identical PCNA expression was observed in untreated endometrial sections (not shown). Scale bar, 200 µm. B, Uterine section from Emx2 transfected mice showing greatly reduced glandular PCNA expression and minimally reduced stromal PCNA. Increased Emx2 reduces glandular proliferation. C, Emx2 transfection reduces endometrial BrdU incorporation. Mice treated with BrdU before they were killed show decreased endometrial BrdU compared with control (Ctl) mice treated with empty vector. *, Significant difference from control (P < 0.001) (n = 3 in each group). D, Ishikawa cells, a well-differentiated human endometrial epithelial adenocarcinoma cell line, were transfected with either control vector (Ctl), Emx2 antisense (AS), or pcDNA3.1/Emx2 (Emx2). The number of days after transfection is shown on the x-axis and log cell number on the y-axis. After 6 d of culture, a discernable difference in cell number was detected. The difference in cell number between treatment groups continued to amplify with further days in culture. Each experiment was performed in quadruplicate.

 
To directly observe the effect of EMX2 on cell proliferation, we transfected the uterine epithelial Ishikawa cell line with either Emx2, Emx2 antisense, or control construct and subsequently observed the effect on cell proliferation and cell number. Cells transfected with the Emx2 expression construct, but not those transfected with control or Emx2 antisense, had diminished cell number. After 10 d, there were approximately 100 times the number of cells counted in the control and antisense group compared with the Emx2 group (P < 0.001). Taken together, these experiments indicate that EMX2 has a significant effect on endometrial epithelial cell proliferation.

Apoptosis was unaltered by increased Emx2 expression. There was no change in activated caspase3 between control mice and those transfected with the Emx2 expression construct. Figure 5Go demonstrates equal levels of activated caspase3 as indicated by darkly stained cells, and light background staining indicates inactive caspase. The effect of Emx2 on endometrial receptivity in murine litter size is likely mediated by its profound effect on endometrial cell proliferation.



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Fig. 5. Emx2 Does Not Alter Uterine Apoptosis

Activated caspase3 was used to detect apoptosis after transfection of the uterus with empty vector (Control) or Emx2 expression vector (Emx2). Activated caspase three stains dark brown. No difference was noted in activated caspase3 between groups. Scale bar, 200 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Emx2 has an essential role in embryogenesis as demonstrated by defects seen after targeted disruption; both the central nervous system and urogenital tract are significantly effected. Emx2 mutant mice die perinatally due to the lack of kidney development. Embryonic patterning genes used during development of the reproductive tract are also often used in reproductive tract remodeling in the adult. We have previously shown that the same Hox genes required for reproductive tract development are also expressed in the adult female reproductive tract where they are necessary for fertility and pregnancy (15, 16, 17, 18). Here we show that the appropriate expression of Emx2 in the adult female reproductive tract is similarly necessary for normal reproductive function. Emx2 expression is normally repressed at a time in the estrous cycle when endometrial remodeling and embryo implantation occur. Overexpression of Emx2 at this time results in impaired reproduction and decreased litter size. Emx2 is essential for development of normal endometrial receptivity to the implanting blastocysts.

Increased Emx2 expression resulted in decreased proliferation of endometrial cells in this model. The normal repression of Emx2 expression at the time of implantation allows for increased proliferation of the endometrial glandular and stromal cells. Concomitant with this increase in proliferation, there was also no change in apoptosis. Markers of differentiation are also not altered by alteration of Emx2. Emx2 is required for regulating the proliferation of cells necessary for implantation without affecting their differentiated phenotype receptivity to the blastocyst. Increased cell number and endometrial tissue volume may provide the necessary infrastructure for optimal embryo implantation. In humans, increasing endometrial thickness correlates with higher implantation rates and is supportive of this conclusion (19). The decreased complement of fully differentiated endometrial cells that develop after augmented Emx2 expression still allows pregnancy but may not permit maximal implantation or normal litter size. Progesterone normally decreases endometrial proliferation in the receptive phase of the estrous cycle. The increased proliferation in response to decreasing Emx2 expression at this phase in the cycle may help counteract the effect of progesterone and allow appropriate tissue remodeling and development of the receptive endometrium.

We have previously shown that, in the human menstrual cycle, EMX2 mRNA is decreased in the luteal phase at the time of implantation. This suggests that the necessity of carefully regulated Emx2 expression, as demonstrated here in murine reproduction, may also be true in human reproduction. EMX2 is directly and negatively regulated by HOXA10 in human endometrial cells (10, 20). HOXA10 expression increases during the luteal phase of the menstrual cycle and in the receptive phase of the murine estrous cycle. Hoxa10 is necessary for endometrial receptivity; Hoxa10(–/–) mice produce normal embryos which are viable in wild-type uterus, but neither the mutant embryos nor wild-type embryos will implant in the Hoxa10-deficient uterus (21). Similarly, blocking maternal adult Hoxa10 expression with antisense diminishes litter size, suggesting that this gene is essential not only for normal urogenital tract development but also for endometrial development in the adult (22). Emx2 and Hoxa10 are each used in both embryonic uterine development and adult remodeling. The expression pattern, function in murine reproduction, and the regulation by a gene know to be required for human implantation all implicate EMX2 in this process in humans.

Alterations in EMX2 expression been associated with disease states in humans. The affected cells often have a change in proliferative phenotype. In endometrial tumors, EMX2 expression is decreased consistent with EMX2’s role in limiting proliferation (12). In contrast, although expression of most genes is decreased in the atrophied endometrium of the postmenopausal woman, EMX2 expression is increased over that of normal cycling women (12). This increase after menopause may prevent unnecessary endometrial proliferation when this organ system is no longer functional. We have previously shown that Emx2 expression is increased in women with endometriosis (13). Endometriosis is a condition in which endometrial glands and stroma develop and continue to proliferate in ectopic extrauterine locations. This condition occurs in 10–15% of reproductive aged women and causes pain and infertility. Alterations in Emx2 may change the proliferative potential of endometrial tissue.

The proliferation-suppressing function of Emx2 parallels that seen in the nervous system, where Emx2 has been shown to be essential for the regulation of appropriate proliferation of the nervous system (23, 24, 25, 26, 27). Emx2 is expressed exclusively in proliferating cells of the reticular zone of the mouse cerebral cortex, suggesting a similar role of proliferation (28). Similarly, use of region-specific molecular markers shows that hippocampus fields are specified and correctly positioned in Emx2 mutants, although they fail to form a morphological gyrus likely due to insufficient proliferation and therefore cell number (29). Emx2 is required for normal growth but not for cell specification. Emx2 is expressed at high levels in adult neural stem cells in vitro and is down-modulated upon differentiation (23). As in the endometrial cell model, overexpression of Emx2 in the stem cells has an antiproliferative effect, yet does not influence a particular differentiation pathway. More recently, Galli et al. (27, 30) have shown that the periventricular region of the adult telencephalon is a neurogenic area that displays a large number of multipotent stem cells. Adult neural stem cells isolated from this region express Emx2 that is down-regulated significantly upon differentiation in neurons and glia. Emx2 is a regulator of self-renewal in neural stem cells. Increasing Emx2 in adult neural stem cells greatly reduces the rate of proliferation Both in the central nervous system and the reproductive tract, Emx2 appears to regulate proliferation independently from differentiation. Appropriate growth and assignment of appropriate cell identity both contribute to development. It is likely that in both brain and reproductive tract, Emx2 allows the independent regulation of these two processes. The targets of Emx2 transcriptional regulation that mediate proliferation await identification. Defective cytoarchitecture seen after Emx2 alteration may be due to reduced numbers of one particular cell type over another. Emx2 has a conserved function in both the central nervous system and the reproductive tract.

This study shows for the first time that adult Emx2 regulation is essential for normal reproductive competence. In the reproductive tract, the increased proliferation afforded by decreased Emx2 in the window of endometrial receptivity likely defines the total endometrial mass and therefore litter size. The diminished endometrial expression of Emx2 seen at the time of embryo implantation is likely necessary for appropriate proliferation, decidualization, and optimization of litter size. Emx2 operates in a conserved pathway that is used in both embryogenesis and adult reproductive function. Proliferation without affecting differentiation or cell identity allows for altered size and cytoarchitecture of individual cell types and tissues essential for development and here is shown to also be essential for optimization of litter size. Emx2 is a proliferation-inhibiting gene essential for mammalian reproduction. Disorders of EMX2 regulation result in disease characterized by abnormal proliferation of uterine endometrium. Elucidation of the role of EMX2 in endometrium may provide opportunities for treatment.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice
Adult reproductive age CD1 mice were obtained from Charles River (Wilmington, MA) and housed in Yale University Animal Care facilities under an approved protocol. All mice were administered food and water ad libitum. In all breeding studies, one to three females were housed with a proven fertile male. The day of vaginal plug detection was designated d 1 of pregnancy, when females were removed from the male and housed in groups of two to five. For analysis of term parameters, pregnant females were checked at 0800 h and 1800 h from d 18 of pregnancy until birth, when gestation length was determined to the half day and litter size recorded. Pups were weighed 6–12 h after birth.

Cell Culture
Ishikawa cells are a well-differentiated endometrial epithelial line of human origin (31, 32, 33, 34, 35, 36). Ishikawa cells were maintained in charcoal stripped, phenol-red free MEM (Invitrogen, Carlsbad, CA) containing 2.0 mM L-glutamine and Earl’s salts, supplemented with 10% FBS, 1% sodium pyruvate, and 1% penicillin /streptomycin.

DNA/Liposome Preparation and Transfection
DNA (2 µl) was mixed with 300 µl Opti-MEM reduced serum medium (Life Technologies, Gaithersburg, MD), added to 10 µl of 22 C liposome [a 3:1 (wt/wt) formulation of 2,3-dioleyloxy-N-[2 (sperminecaboxamido)ethyl]-N-N-dimethyl-1-propanaminium trifluoroacetate (DOPSA) and dioleoylphosphatidyl ethanolamine (DOPE) (Life Technologies)] in 300 µl 37 C OptiMEM and incubated for 45 min at room temperature. A final concentration of 16 µl/ml DNA and 40 µg/ml liposome was obtained by dilution with 1x Dulbecco’s PBS (Life Technologies) to a total volume of 100 µl. Transfer to each uterine horn was performed as previously described (22).

Emx2 Constructs
Emx2 cDNA was a generous gift of R. Galli (27). Emx2 cDNA was subcloned into pcDNA 3.1. Empty pcDNA 3.1 vector was used as a control. Emx2 antisense was a phosphothiorate-modified 30-base antisense oligonucleotide that targeted the start of Emx2 translation.

Immunohistochemistry
Cellular expression of calcitonin, prolactin, IGFBP-1 and proliferating cell nuclear antigen (PCNA) and caspase-3 were evaluated by immunohistochemistry using rabbit antibodies to calcitonin (AO 576: Dako, Carpinteria, CA), prolactin (AO 569: Dako), activated caspase-3 (Cell Signaling) and PCNA (Fl-2b1 Santa Cruz), respectively. Briefly, four full-thickness biopsies were obtained at the time of histological evaluation from each uterine horn. The specimens were embedded in paraffin and serial 5-mm sections obtained. The sections were deparaffinized in xylene and ethanol. Endogenous peroxidase was blocked with 3% H2O2. After a 45-min incubation with 1.5% normal goat blocking serum, the sections were incubated overnight at 4 C with primary antibody (calcitonin 1:2000; prolactin 1:800, PCNA 1:400). The sections were then incubated with biotinylated goat antirabbit secondary antisera for 60 min, avidin and biotinylated peroxidase (Vectastain; Vector Laboratories, Burlingame, CA) for 45 min and diaminobenzidene (400 mg/ml) for 5 min. Hematoxylin and Li2CO3 were used for counterstaining. One hundred consecutive cells in five nonadjacent microscopic fields were evaluated for staining to quantify the expression of each cell-type specific marker.

BrdU Labeling
Mice were injected ip with BrdU 50 µg/g on the 2 d before they were killed. All BrdU-treated mice had been treated with Emx2 constructs as described above on d 2 of pregnancy. Euthanasia was accomplished by CO2 narcosis. BrdU labeling was visualized using a monoclonal anti-BrdU antibody (Roche Diagnostics Corp., Indianapolis, IN). BrdU-labeled cells were counted by two individuals blinded to the treatment regimen. Cells were visualized with a x40 objective and a total of at least 1000 cells were counted from each animal. The mean percentage of positive cells is reported. Each group consisted of three animals.

Cell Proliferation Assays
Ishikawa cells were transfected with the Emx2 containing plasmid described above, empty vector or Emx2 antisense. Cell proliferation assay was performed as previously described (37). Briefly, cells were plated at a density of 105 cells/chamber and incubated for 48 h. Cells were counted on the undersides of filters after fixation and staining with crystal violet. Each experiment was performed in quadruplicate.


    FOOTNOTES
 
First Published Online June 30, 2005

Abbreviations: BrdU, 5'-Bromo-2' deoxyuridine; ems, empty spiracles; IGFBP1, IGF binding protein-1; IHC, immunohistochemistry; PCNA, proliferating cell nuclear antigen.

Received for publication March 18, 2005. Accepted for publication June 21, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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