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a Department of Animal and Dairy Sciences, Auburn University, Alabama 36849-5415
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-positive (ER+) phenotype by, and increased DNA synthesis in, nascent glandular epithelium (GE). To determine whether ER activation is required for this process, gilts were treated daily with either vehicle, the antiestrogen ICI 182,780 (ICI), estradiol-17ß valerate (EV), or both ICI and EV. Treatments began on PND 0, before onset of adenogenesis, or on PND 7, after onset of gland proliferation. Uteri obtained on PNDs 7 and 14 (study one) or on PND 14 (study two) were weighed; uterine histology was evaluated; DNA synthesis in luminal epithelium and GE was characterized by determining 5-bromo-2'-deoxyuridine (BrdU) labeling index; and patterns of ER mRNA expression were evaluated in situ (study one). Gland genesis was inhibited by ICI, which decreased gland penetration depth by PND 14 in study one, both endometrial thickness and BrdU-labeling index in GE in study two, and increased stromal cell compaction in both studies. Uterotropic effects of EV included increased gland development and epithelial BrdU labeling and decreased stromal compaction. These effects were inhibited by coadministration of ICI. Treatments did not alter ER mRNA expression, which remained limited to stroma and GE. Data indicate that endometrial maturation and adenogenesis in the neonatal pig require expression and activation of a functional ER system.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to alterations in proliferative behavior, genesis of endometrial glands in porcine [2] and rodent [9–11] uteri involves coordinated changes in epithelial phenotype marked by expression of estrogen receptor-positive (ER+) character in nascent GE [2]. The porcine endometrium is ER negative (ER-) at birth [2]. However, endometrial stroma and GE display ER+ character by PND 15, while luminal epithelium (LE) remains ER- [2]. These relationships suggest a role for the ER in genesis of endometrial glands.
Mechanistically, the ER can be activated through estrogen-dependent, genomic effects [12] or through estrogen-independent ER-coupled pathways [13]. Both types of ER-dependent signaling can be attenuated or inactivated with the specific antiestrogen ICI 182,780 (ICI), a 7-alkylamide analogue of estradiol-17ß [14, 15]. In either case, activation or inactivation, tissue response to estrogen or ICI should reflect the distribution of and functional relationships between resident ER+ cells.
Porcine uterine development can be advanced in an age-specific manner by targeted exposure of gilts to estrogen [16]. Estradiol-17ß valerate (EV), administered for 7 days prior to hysterectomy of gilts on PND 7 or 14, increased uterine weight and endometrial thickness, advanced development of uterine glands, and affected patterns of in vitro uterine protein production [16]. Effects of EV were consistently uterotropic and more pronounced when exposure occurred during the proliferative period. Period-specific responses of neonatal porcine uterine tissues to estrogen were interpreted to reflect development of ER+ character by stromal and glandular epithelial cells, which occurs between birth and PND 15 [2, 16]. Data were also interpreted to suggest that ER activation is required for normal uterine growth and endometrial development during the first 2 wk of postnatal life in the pig [2]. If so, then attenuation of ER activity should be antiuterotropic and retard or inhibit endometrial adenogenesis.
Here, EV and ICI were used as tools with which to stimulate or inhibit ER activity in the pig between birth and PND 14 in order to test the hypothesis that ER activation is required for normal endometrial development and adenogenesis in the neonatal porcine uterus. Objectives were to determine effects of ICI and EV, administered through infantile and proliferative periods (PNDs 0–13) of endometrial development (study one), or during the proliferative period alone (PNDs 7–13; study two), on patterns of uterine growth, endometrial development, adenogenesis, and epithelial DNA synthesis as determined on PND 7 and/or PND 14. Effects of age and treatment from birth on patterns of ER mRNA expression were also determined by in situ hybridization (ISH).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crossbred gilts were assigned to treatment groups at birth (PND 0). In study one, gilts (n = 7–11 per group) received either corn oil vehicle (CO), ICI (125 µg/kg BW per day, s.c.), EV (50 µg/kg BW per day, i.m.), or both ICI and EV (IE) daily from PND 0 through either PND 6 or PND 13. Dose and route of administration of ICI were derived from data for in vivo responses of immature and adult rats and ovariectomized adult pigtail macaques [15]. The ICI dosage was approximately equivalent, on a molar basis, to that of EV. Uteri were obtained with gilts under halothane anesthesia on either PND 7 or 14. Thus, in study one, uteri were obtained from gilts that were either exposed or unexposed to ICI and/or EV from birth throughout the infantile (PNDs 0–6) and proliferative (PNDs 7–13) periods of neonatal endometrial development. In study two, gilts (n = 7–8 per group) assigned to treatment groups at birth received either CO, ICI, EV, or IE daily from PND 7 through PND 13, during the proliferative period of neonatal endometrial development only. Uteri were then obtained on PND 14. All procedures involving animals were approved by the Auburn University Institutional Animal Care and Use Committee and were in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [17].
Tissue Processing and Histomorphometry
Connective tissue, cervix, oviducts, and ovaries were trimmed from each uterus, and uterine wet weight was recorded. A segment from the middle of each uterine horn was fixed in 4% paraformaldehyde, embedded in Paraplast-Plus (Sherwood Medical, St. Louis, MO), and sectioned at 5–6 µm for histological evaluation. Tissues were stained with buffered azure eosin (study one) or hematoxylin and eosin (study two) for light microscopy. Endometrial thickness was measured, using a calibrated ocular micrometer, as the distance (µm) from the basal lamina of the LE to the border of the endometrial stroma and the inner circular myometrium. Endometrial gland penetration depth was measured from the basal aspect of the LE, immediately adjacent to the mouth or opening of the gland into the uterine lumen, to the interface of the basal aspect of the epithelium at the bottom of each gland with the subadjacent stroma. Stromal cell nuclear density was determined for two zones of endometrium, as defined previously [8]. These included the shallow stratum compactum (subadjacent to LE) and deep stratum spongiosum (adjacent to myometrium). Stromal cell nuclei were counted at a magnification of x100 using an ocular grid. Reticle dimensions and a stage micrometer were used to convert data to stromal nuclei per square millimeter.
Metabolic Labeling with BrdU and Determination of BrdU-Labeling Index
Immediately after hysterectomy, 250 mg of tissue from each uterus was placed in 4 ml of phenol-red free Basal Medium (BME; Gibco BRL, Gaithersburg, MD) containing 0.4 mM 5-bromo-2'-deoxyuridine (BrdU) and incubated in a shaking water bath (37°C, 4 h) under an atmosphere of 5% CO2:95% air. Labeled tissue was then fixed in Carnoy's fluid for 2 h [18] and embedded in Paraplast-Plus. To visualize BrdU-labeled cells, tissue sections (5–6 µm) were treated with 2 N HCl at 37°C for 15 min [19]. BrdU-positive nuclei were then detected using the BioStain Super ABC kit (Biomeda, Foster City, CA) and a mouse anti-BrdU monoclonal antibody (mAb, BU-33; Sigma Chemical Co., St. Louis, MO). Anti-BrdU (BU-33) was diluted in PBS (pH 7.4 with 1% BSA and 0.05% Tween 20) with 3% normal goat serum added and used at a concentration of 1 µg/ml. Negative control sections received irrelevant mouse IgG (1 µg/ml) in place of anti-BrdU mAb. BrdU-labeling index was determined for LE and for GE. For each area, at least 1000 cells (labeled and unlabeled) were counted. Labeling index was expressed as the percentage of total cells counted that contained BrdU-labeled nuclei.
ISH
ISH procedures were identical to those described previously [2]. ER mRNA was localized by ISH [20] in paraformaldehyde-fixed uterine tissues. Antisense and sense [35S]UTP-labeled (1000 Ci/mmol; ICN, Costa Mesa, CA) cRNA probes were produced by in vitro transcription from the EcoRI- and BamHI-linearized oER8 cDNA template ([21]; a gift from Dr. Nancy H. Ing) using a MaxiScript kit (Ambion, Austin, TX). The RNA polymerases T7 and SP6 were used to transcribe antisense and sense probes, respectively. Complementary RNA probes produced from the oER8 cDNA used here were validated previously for detection of ER in pigs [2]. Antisense and sense sections were included on each slide, and all slides were processed together. Autoradiography was accomplished using NTB-2 emulsion (Eastman Kodak, Rochester, NY), with 8-wk exposure at 4°C. Images representative of ISH results were captured digitally by darkfield microscopy using constant conditions to ensure accurate comparisons between images, as described previously [2].
Statistical Analyses
Porcine neonatal uterine histomorphometric measurements are highly repeatable [22, 23]. Therefore, except for gland penetration depth and stromal cell nuclear density analyses in study two (which were analyzed first to determine that tissue section and microscopic field within section were not significant sources of variation), single cross sections were measured for each animal in the present studies. Endometrial thickness and gland penetration depth data were pooled for dorsal and ventral locations, and this data set was used in statistical analyses for these responses. Data for uterine weight, endometrial thickness, gland penetration depth, stromal cell nuclear density, and BrdU-labeling indices were subjected to least-squares ANOVA using General Linear Models procedures [24]. Histomorphometric data (endometrial thickness and gland penetration depth) were log transformed prior to analysis. Statistical models included the main effects of age (PND 7, PND 14; study one only), treatment (CO, ICI, EV, IE), their interactions, and animal nested within treatment by age, as appropriate. For histomorphometric data, uterine wall location (mesometrial and antimesometrial vs. dorsal and ventral) was included in the model, along with uterine wall location by main effect interactions. Tests of significance were based on expectations of the error mean squares. Preplanned comparisons used to determine treatment effects on a within-day basis were CO vs. ICI, CO vs. EV, and EV vs. IE. Data are presented as least-squares means of untransformed values with SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Data for uterine wet weight are presented in Figure 1. When treatments were initiated at birth (study one), effects of treatment, day, and a treatment-by-day interaction were observed (p < 0.001). Normal uterine growth, observed in CO-treated gilts, was reflected by an increase in uterine wet weight from PND 7 to PND 14 (0.53 vs. 1.02 ± 0.18 g; day, p < 0.0001). Uterine wet weight was not affected by ICI administered from birth (study one; Fig. 1A) or from PND 7 (study two; Fig. 1B). Uteri from EV gilts weighed more than those from CO-treated cohorts at each time point in both studies (PND 7, p < 0.10; PND 14, p < 0.01). In study one, administration of ICI with EV from birth (group IE) attenuated uterotropic effects of EV on both PND 7 (p < 0.10) and PND 14 (p < 0.03). Uterine wet weight in IE gilts was also reduced in comparison with that of EV gilts when treatment began on PND 7 (study two; p < 0.01).
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Histology
Photomicrographs depicting uterine histology observed in CO, ICI, EV, and IE gilts treated from birth (study one) or during the proliferative period only (study two) are presented in Figures 2 and 3. Normal endometrial development was observed in CO gilts. GE was observed on PND 7, and glandularity increased between PNDs 7 and 14, when coiled tubular glands were distributed regularly throughout the endometrial stroma (Fig. 2). Uterine histology on PND 14 was similar for CO gilts in the two studies (Fig. 2 vs. Fig. 3; D14 CO).
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Treatment of gilts with ICI produced marked antiuterotropic effects as reflected by endometrial histology. In study one, uteri obtained on PND 7 from ICI gilts contained endometrial glands that were less consistently well developed than those in uteri obtained from CO gilts on the same day (Fig. 2; D7 CO vs. D7 ICI). However, effects of ICI were most pronounced by PND 14, when endometrial gland development was dramatically retarded by comparison with tissues obtained from CO-treated gilts on that day (Fig. 2; D14 CO vs. D14 ICI). Endometrial gland development was also retarded to some extent when treatment with ICI was limited to the proliferative period (study two, Fig. 3; D14 CO vs. D14 ICI). However, uterine gland development in these gilts was still more advanced than in gilts treated with ICI from birth (Fig. 2, D14 ICI vs. Fig. 3, D14 ICI).
As expected, EV treatment induced precocious endometrial development. Maximal endometrial development was observed in EV-treated gilts on PND 14 in study one (Fig. 2; D14 EV), when glands had proliferated throughout the stroma to the myometrium and precocious endometrial folds were evident. Similarly, treatment with EV for 7 days prior to PND 14 (study two; Fig. 3, D14 EV) caused precocious endometrial gland proliferation throughout the stroma. Treatment with ICI attenuated EV-induced growth of the endometrium, with an intermediate level of gland development observed on all days in each study for IE gilts (Figs. 2 and 3; EV vs. IE groups).
Morphometric Data
Data for endometrial thickness are presented in Figure 4. In study one, endometrial thickness increased from PNDs 7 to 14 (p < 0.001), and a treatment-by-day interaction was detected (p < 0.07). When treatment began at birth, ICI did not affect endometrial thickness on PND 7 or PND 14 (Fig. 4A). However, treatment with ICI from PNDs 7 to 13 (study two, Fig. 4B) reduced endometrial thickness by PND 14 (p < 0.05). Uteri from all gilts treated with EV displayed increased endometrial thickness relative to CO gilts (p < 0.01). When ICI was administered with EV from birth (IE), uterotropic effects of EV on endometrial thickness were ablated (p < 0.0001) on PND 7 but were only attenuated (p < 0.02) on PND 14 (Fig. 4A). Similarly, coadministration of ICI and EV from PNDs 7 to 13 attenuated (p < 0.01), but did not completely ablate, uterotropic EV effects (Fig. 4B).
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Data for stromal cell nuclear density in the stratum compactum (shallow endometrial stroma) and the stratum spongiosum (deep endometrial stroma) are presented in Figure 5. This response was interpreted to be an estimate of the relative density of stromal fibroblasts per unit of endometrial area. Overall, in study one, stromal cell nuclear density decreased from PND 7 to PND 14 in the stratum spongiosum (day p < 0.0001). Significant treatment-by-day interactions were observed for this response in both the stratum compactum (p < 0.05) and the stratum spongiosum (p < 0.01). Treatment with ICI from birth increased stromal cell nuclear density on PND 7 (p < 0.01) in the stratum compactum but did not affect the deep stratum spongiosum. Data reflected the more compact shallow endometrium observed histologically for ICI- as compared to CO-treated gilts in these groups (Fig. 2). More prolonged treatment with ICI from birth did not affect stromal cell nuclear density in either endometrial zone on PND 14 (Fig. 5, A and B). However, when ICI administration was confined to the period from PNDs 7 to 13, stromal cell nuclear density increased (p < 0.04) in the stratum spongiosum (Fig. 5D). EV decreased stromal cell nuclear density in both areas of the endometrium on PND 7 and PND 14 in study one (p < 0.001) and on PND 14 in study two (p < 0.01). Coadministration of ICI with EV attenuated EV effects in both stromal zones on PND 7 (p < 0.01). However, antiestrogenic effects of ICI on stromal cell nuclear density were confined to the stratum compactum on PND 14 (study one; p < 0.07). Coadministration of ICI with EV from PNDs 7 to 13 did not alter the EV-induced decrease in stromal cell nuclear density observed on PND 14 in study two (EV vs. IE; p > 0.20).
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Data for endometrial gland penetration depth are presented in Figure 6. In study one, endometrial gland penetration depth increased from PND 7 to PND 14 (p < 0.001), and a treatment-by-day interaction was observed (p < 0.01; Fig. 6A). Given alone, ICI administered from birth did not affect endometrial gland penetration depth on PND 7. However, when given from PNDs 0 to 13, ICI reduced (p < 0.02) gland penetration depth markedly by PND 14. In contrast, gland penetration depth was not affected (p < 0.13) when animals were given ICI from PNDs 7 to 13 (Fig. 6B). Uteri from gilts treated with EV displayed increased gland penetration relative to CO uteri in both studies on both days (p < 0.05). This EV response was ablated on PND 7 in IE gilts (study one; p < 0.02), and was attenuated (p < 0.001) by PND 14 in IE gilts from both studies.
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Epithelial BrdU Labeling
Representative immunostaining results for BrdU labeling are presented in Figure 7. Results of epithelial BrdU-labeling index determinations are presented in Figure 8. Overall, in study one, no effect of day was observed for this response in either LE or GE. However, treatment-by-day interactions were detected for BrdU-labeling index in both LE (p < 0.06) and GE (p < 0.03). In both studies, BrdU-labeling index was lower (p < 0.01) in LE than in GE. When treatments began at birth, BrdU-labeling index for LE did not differ between CO- and ICI-treated gilts on PND 7 (Fig. 8, A and C) but increased (p < 0.05) in ICI-treated gilts by PND 14 (Fig. 8A). In study two (Fig. 8C), administration of ICI from PNDs 7 to 13 did not affect BrdU-labeling index in LE on PND 14. Administration of EV from birth increased BrdU-labeling index in LE on both PND 7 and PND 14 in study one (Fig. 8A; p < 0.05) and on PND 14 in study two (Fig. 8C; p < 0.01). Coadministration of ICI with EV ablated LE response to EV on PND 7; it attenuated the response on PND 14 in study one (Fig. 8A; p < 0.01) and on PND 14 in study two (Fig. 8C; p < 0.05).
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Glandular epithelial BrdU-labeling index (Fig. 8, B and D) was not affected by ICI treatment from birth (Fig. 8B) but was reduced (p < 0.01) on PND 14 when ICI was administered only from PNDs 7 to 13 (Fig. 8D). Administration of EV to neonatal gilts from birth increased BrdU-labeling index in GE on PND 7 (p < 0.01) but not on PND 14 (Fig. 8B). In contrast, treatment with EV from PNDs 7 to 13 increased (p < 0.01) BrdU-labeling index in GE on PND 14 (Fig. 8D). Coadministration of ICI with EV from birth in study one attenuated (p < 0.01) positive EV effects on BrdU-labeling index in GE on PND 7 but not on PND 14 (study one; Fig. 8B). Coadministration of ICI from PNDs 7 to 13 abolished this GE response to EV in study two (Fig. 8D; p < 0.01).
ISH for ER mRNA
Representative antisense and sense background images depicting specific in situ localization of ER mRNA in a neonatal porcine uterus are presented in Figure 9. Endometrial ER mRNA localization in uteri from animals treated from birth (study one) is illustrated in Figure 10. Positive ER mRNA signal was detected in stroma and nascent GE of the normal PND 7 uteri (D7 CO). On PND 14, stroma remained ER+, and GE was intensely ER+ in uteri from CO-treated gilts, while LE remained ER-. Treatments (CO, ICI, EV, and IE) did not affect spatial expression patterns of ER mRNA, or apparent signal intensities, on either PND 7 or 14.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ontogeny of endometrial ER expression, like uterine gland genesis, is also a postnatal event in the pig [2]. Whereas ER is undetectable by ISH at birth [2], evidence of ER mRNA in both GE and endometrial stroma, but not in LE, on both PNDs 7 and 14 in CO gilts reinforces earlier data interpreted to indicate that ER is a marker of GE differentiation in the porcine uterus [2]. Given that spatial patterns of endometrial ER mRNA expression are mirrored by nuclear staining patterns for ER protein in the neonatal porcine uterus [2, 25], present data can be interpreted to indicate that stromal and glandular epithelial cells with potentially functional ER are present in the porcine endometrium by at least PND 7.
To show that a specific gene product is involved mechanistically in organogenesis, expression of the gene in question must be correct, both temporally and spatially, and disruption of gene expression, or inhibition of the function of the gene product, must prevent or retard the normal organogenetic process [26]. Previous studies [2], reinforced by current data, clearly establish a temporal and spatial link between ER expression patterns and onset of endometrial adenogenesis in the pig. At present, gene knockout technology is not possible in the pig. Therefore, ICI, a steroidal antiestrogen and inhibitor of ER function (see below), was employed to test the hypothesis that ER activation is required for normal uterine gland development in the neonatal pig.
In both study one (in which treatments were administered to neonatal gilts from birth, prior to acquisition of ER+ character by endometrial cells and onset of uterine adenogenesis), and study two (in which treatments were administered during the proliferative period of endometrial development, after onset of events associated with adenogenesis), ICI was consistently antiadenotrophic. However, morphogenetic effects of ICI, like those of EV, were specific to the developmental periods during which treatments were administered.
In study one, specific antiadenotrophic effects of ICI were clear, progressive, and most pronounced by PND 14, when gland development was markedly retarded. When ICI was administered from birth, ICI-induced inhibition of gland development was not associated with reduced endometrial thickness or BrdU-labeling index in GE, but it was associated with increased stromal cell density in the shallow stratum compactum on PND 7. In contrast, ICI administered during the period from PNDs 7 to 13 (study two) did not reduce mean gland penetration depth but did reduce overall endometrial thickness through effects on stromal cell organization, as reflected by increased density of nuclei per unit area in the deep stratum spongiosum, and did decrease BrdU-labeling index in GE. To the extent that effects of ICI 182,780 can be interpreted to reflect specific disruption of ER function [12], antiadenotrophic effects of ICI observed here provide pharmacological evidence that ER activation is required for normal development of uterine glands and endometrial maturation in the neonatal pig. Thus, neonatal porcine endometrial development is ER dependent.
Differences in tissue and cell responses observed between studies one and two are consistent with the idea that ER-dependent cell behaviors associated with normal endometrial organization change over time between birth and PND 14. Both antiuterotrophic effects of ICI and uterotropic effects of EV were expected to reflect the relative presence and changing distribution of ER in situ [16]. In this regard, consideration must also be given to the fact that signaling through the ER can originate directly, via estrogen-dependent activation [12], or indirectly, through cross-talk between membrane-mediated signal transduction systems and the ER [13]. It is not likely that the neonatal porcine uterus is exposed to more than negligible levels of circulating estrogens [27, 28]. Similarly, it does not seem likely, given probable limitations of substrate, that the neonatal endometrium might acquire the ability to synthesize estrogen, although aromatase expression is documented for the porcine endometrium [29, 30]. However, locally produced growth factors or cytokines could activate the ER system in the early postnatal uterus and affect the behavior of target cells [13, 31, 32]. The possibility that ER+GE cells may be more responsive to the paracrine action of locally produced growth factors such as epidermal growth factor or insulin-like growth factor-I was suggested earlier [2, 33]. It is likely, of course, that growth factors and other paracrine effectors of gland development are acting through their cognate receptors, independently of the ER system in the developing endometrium. Nevertheless, antiuterotrophic effects of ICI indicate that the ER system is functioning at some level in the neonatal porcine uterus to support endometrial remodeling.
Present data indicating that neither patterns of expression nor relative signal intensities observed for ER mRNA in situ were affected by ICI treatment from birth are generally consistent with ICI-induced disruption of ER function via alterations in ER protein turnover and stability, without reductions in cellular ER protein levels [34] or changes in steady state levels of ER mRNA [35]. Thus, inhibition of gland genesis and alterations in uterine wall organization induced by ICI here are likely due to inhibition of ER function, rather than reduced ER expression. Addition of ICI 164,384 to polarized rat uterine epithelial cell cultures altered vectoral protein secretion in a manner opposite to that observed for estradiol [36]. Also, antiestrogens, including the ICI compounds, can act through ER-independent mechanisms [37–40]. Collectively, observations can be interpreted to indicate that, in addition to disruption of ER function, the antiadenotrophic effects of ICI reported here could reflect changes in vectoral patterns of epithelial secretion and associated alterations in tissue microenvironment required to ensure morphogenetically critical stromal-epithelial interactions that support normal endometrial maturation [41–43].
Current results confirm previous observations [16] indicating that EV is uterotropic in the neonatal pig and, generally, that uterine responses to estrogen are specific to exposure period, tending to increase with postnatal age. Trophic effects of estrogen included increased uterine wet weight, endometrial thickness, and glandularity, all evidence of precocious endometrial maturation. The lack of a consistently evident stratum compactum in the shallow endometrial stroma after treatment of gilts with EV led Spencer et al. to propose that estrogen causes stromal disorganization in the neonatal porcine uterus [16]. Dramatic decreases in density of stromal nuclei per unit area in both shallow and deep stroma in all EV-treated gilts, on all days, provide a characterization of EV-induced stromal disorganization in the porcine endometrium. The mechanisms of this disorganization are not known at present but could include changes in the quality of the extracellular matrix and changes in the orientation and size of uterine mesenchymal cells [44, 45].
Treatment effects on BrdU-labeling index were epithelial compartment-specific and were related to both duration and period (infantile vs. proliferative) of exposure to estrogen. In both studies one and two, EV increased BrdU-labeling index in LE. Results of current and previous studies [2] indicate that LE remains essentially ER- between birth and PND 14 in the neonatal gilt, and that estrogen exposure from birth does not induce premature ER expression in LE during this period. Therefore, positive effects of EV on BrdU-labeling index observed for LE, as well as antagonistic effects of ICI observed for this response in IE-treated gilts, probably reflect indirect actions of estrogen and antiestrogen. These agents are likely to be operating through ER+ stroma to alter the relative production and/or effectiveness of paracrine-acting mediators of LE proliferation. Estrogen-induced, ER-dependent, stromal induction of ER- epithelial proliferation was demonstrated elegantly for the murine uterus [43, 46].
In the neonatal pig, GE differentiates from LE during the first week of postnatal life [1, 2]. Current and previous observations [2] establish that this event is marked by development of ER+ character in stromal cells and nascent GE, which are both ER- at birth [2]. Whether stromal ER expression precedes or accompanies differentiation of ER+GE remains to be determined for the neonatal gilt. It will be important to define precise temporal and spatial patterns of endometrial ER expression during early neonatal life in order to determine whether mechanisms of estrogen and antiestrogen action on epithelial cells are direct or stromally mediated.
Adenogenesis is defined here as the process by which endometrial glands develop. Cell behaviors required for this process include proliferation, migration, and both cell-cell and cell-matrix interactions that support tissue remodeling [1]. Overall, present data indicate that ICI inhibited and EV stimulated adenogenesis in the neonatal porcine uterus. Treatment effects, particularly for BrdU-labeling index, a measure of cell proliferation, reflected both period and duration of exposure. Administered from birth, neither ICI nor EV affected BrdU-labeling index in GE on PND 14. In contrast, when administered from PND 7, ICI reduced and EV increased the proportion of GE found to be actively synthesizing DNA on PND 14. These differences in BrdU-labeling index for GE may reflect the fact that treatments began prior to GE differentiation and rapid GE proliferation in study one, but after GE differentiation, during peak GE proliferation in study two. Thus, epithelial cells were at a different stage of maturity and, potentially, in proportionately different stages of the cell cycle when first exposed to ICI and/or EV in the two studies. Relative proliferative effects of steroids are recognized to be affected by the cell cycle status of receptor-positive cells [47].
In general, coadministration of ICI decreased tissue responses to EV. Interestingly, however, coadministration of ICI with EV ablated many responses by PND 7 but only attenuated such responses by PND 14. Thus, the extent to which a constant dose of ICI was sufficient to ablate specific EV-induced uterine responses, including endometrial thickness, gland penetration depth, and BrdU-labeling index for LE, was inversely related to duration of treatment from birth. Such differential effects of treatment may reflect the level of dependence of specific endometrial remodeling events on the ER and point to the possibility that other factors in addition to direct ER activation may be contributing to this process during the second postnatal week.
In summary, present data confirm that EV is acutely uterotropic and establish that ICI 182,780 is antiuterotrophic in the neonatal gilt. Observations can be interpreted to indicate that an activated ER system is required for normal endometrial adenogenesis in the neonatal porcine uterus. Treatments did not affect temporal or spatial patterns of endometrial ER expression at the mRNA level, which was confined to stroma and nascent GE in all cases. Thus, both normal and treatment-associated effects, documented here for the neonatal porcine endometrium, are likely to reflect alterations in stromal-epithelial interactions and associated changes in local tissue microenvironments, whether normal or aberrant, that are specifically uterotropic or antiuterotrophic and that support specific types of remodeling and differentiative events [1, 25]. This model system can now be exploited to identify paracrine effectors of normal and aberrant endometrial development, as well as extracellular matrix conditions that affect cell behaviors necessary for endometrial gland development and cytodifferentiation in the pig. Also of interest are effects of transient neonatal steroid exposure on subsequent uterine function [1], especially in light of reports that such exposure to estrogens and related xenobiotics can alter subsequent uterine gene expression patterns and responses to steroids permanently [48–50].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 334 844 1519; fbartol{at}acesag.auburn.edu
Accepted: February 9, 1999.
Received: November 9, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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knockout mouse. Biol Reprod 1998; 59:470–475.This article has been cited by other articles:
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 B. Berghofer, T. Frommer, G. Haley, L. Fink, G. Bein, and H. Hackstein TLR7 Ligands Induce Higher IFN-{alpha} Production in Females J. Immunol., August 15, 2006; 177(4): 2088 - 2096. [Abstract] [Full Text] [PDF]
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W. Yan, P. L Ryan, F. F Bartol, and C. A Bagnell Uterotrophic effects of relaxin related to age and estrogen receptor activation in neonatal pigs. Reproduction, May 1, 2006; 131(5): 943 - 950. [Abstract] [Full Text] [PDF]
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 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]
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K. Hayashi and T. E. Spencer Estrogen Disruption of Neonatal Ovine Uterine Development: Effects on Gene Expression Assessed by Suppression Subtraction Hybridization Biol Reprod, October 1, 2005; 73(4): 752 - 760. [Abstract] [Full Text] [PDF]
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 Y. Yin and L. Ma Development of the Mammalian Female Reproductive Tract J. Biochem., June 1, 2005; 137(6): 677 - 683. [Abstract] [Full Text] [PDF]
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C. A Oliveira, G. A B Mahecha, K. Carnes, G. S Prins, P. T K Saunders, L. R Franca, and R. A Hess Differential hormonal regulation of estrogen receptors ER{alpha} and ER{beta} and androgen receptor expression in rat efferent ductules Reproduction, July 1, 2004; 128(1): 73 - 86. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D. Carpenter, and T. E. Spencer Neonatal Estrogen Exposure Disrupts Uterine Development in the Postnatal Sheep Endocrinology, July 1, 2004; 145(7): 3247 - 3257. [Abstract] [Full Text] [PDF]
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 T. E. Spencer and F. W. Bazer Uterine and placental factors regulating conceptus growth in domestic animals J Anim Sci, January 1, 2004; 82(13_suppl): E4 - 13. [Abstract] [Full Text] [PDF]
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K. D. Carpenter, C. A. Gray, T. M. Bryan, T. H. Welsh Jr., and T. E. Spencer Estrogen and Antiestrogen Effects on Neonatal Ovine Uterine Development Biol Reprod, August 1, 2003; 69(2): 708 - 717. [Abstract] |
