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BOR - Papers in Press, published online ahead of print October 30, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.011346
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BIOLOGY OF REPRODUCTION 68, 1387–1393 (2003)
DOI: 10.1095/biolreprod.102.011346
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Estrogen-Induced Disruption of Neonatal Porcine Uterine Development Alters Adult Uterine Function1

Becky J. Tarleton3,a,c, Tim D. Bradenb,c, Anne A. Wileya,c, and Frank F. Bartol2,a,b

a Departments of Animal Sciences b Anatomy, Physiology, & Pharmacology, c Cellular and Molecular Biosciences Program, Auburn University, Auburn, Alabama 36849-5415


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the pig, estradiol-17ß valerate (EV) exposure from birth (Postnatal Day [PND] 0) disrupts estrogen receptor-{alpha} (ER)-dependent uterine development and increases embryo mortality in adults. To determine effects of neonatal EV exposure on adult uterine morphology and function, 36 gilts received corn oil (CO) or EV from PND 0 to PND 13. Cyclic and pregnant (PX) adults from each treatment group were hysterectomized on Day 12 after estrus/mating. Treatment and pregnancy effects were determined for uterine weight and horn volume, uterine luminal fluid (ULF) protein and estradiol content, endometrial incorporation of 3H-leucine (3H-Leu) into nondialyzable product, and endometrial mRNA levels for ER, progesterone receptor (PR), uteroferrin (UF), retinol-binding protein (RBP), and keratinocyte growth factor (KGF). Adults cycled normally and had similar numbers of corpora lutea. Uteri of PX gilts contained tubular/filamentous conceptuses, and ULF estradiol content was unaffected by treatment. However, pregnancy increased uterine weight and size only in CO gilts (Treatment x Status, P < 0.01). Treatment reduced ULF protein content (P < 0.01), endometrial 3H-Leu incorporation (P < 0.05), and the pregnancy-associated increase in ULF protein (Treatment x Status, P < 0.01). Treatment did not affect endometrial ER or PR mRNA levels but attenuated the pregnancy-associated increase in UF mRNA (Treatment x Status; P < 0.01), increased RBP (P < 0.10), and decreased KGF mRNA levels (P < 0.05). These results establish that transient postnatal estrogen exposure affects porcine uterine responsiveness to potentially embryotrophic signals and that estrogen-sensitive postnatal uterine organizational events are determinants of uterine size and functionality.

developmental biology, estradiol, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the pig (Sus scrofa), uterine development is initiated prenatally and completed postnatally [1–6]. Postnatal uterine histogenesis involves proliferation of nascent glandular epithelium (GE) that is accompanied by expression of estrogen receptor {alpha} (ER) in both endometrial stroma and GE. Estradiol-17ß valerate (EV) can be used to alter the normal course of endometrial development, as reflected by general uterine hyperplasia, GE proliferation, and precocious development of endometrial folds in neonatal gilts when administered from as early as birth (Postnatal Day [PND] 0) [1, 5, 7]. In contrast, treatment of neonatal gilts with the specific antiestrogen ICI 182,780 from PND 0 through PND 13 inhibited endometrial growth and retarded gland development [7]. Together, these observations were interpreted to indicate that ER is both a marker and a mediator of normal endometrial maturation in the neonatal pig [7].

Data from several species [1, 8, 9] showing that uterine growth and development proceed normally for a period of time following ovariectomy and/or adrenalectomy at birth, were interpreted to indicate that early postnatal uterine development may not require systemic steroid support. However, neonatal uterine tissues are steroid sensitive, and even transient disruption of the normal developmental program by exposure of neonates to steroid hormones or related xenobiotics can have long-term consequences for uterine function and reproductive health [1, 8, 10–14].

Recent data for the pig support the concept that transient perinatal exposure to estrogenic xenobiotics can affect reproductive tract biology later in life. Treatment of gilts with EV for 14-day periods during Postnatal Weeks 1 and 2 or 6 and 7 decreased prepubertal uterine size and altered patterns of protein synthesis and steady-state levels of endometrial mRNA in a manner related to the period when EV exposure occurred and to the age or physiological condition of the animal at the time that tissues were collected [15]. Functionally, EV exposure from PND 0 through PND 13 did not affect ovulation rate in adult gilts but did reduce embryo survival on Gestational Day 45 [1]. These data are consistent with the idea that estrogen-induced disruption of ER-dependent porcine neonatal endometrial development compromises the ability of the adult uterus to respond optimally in support of pregnancy and conceptus development. The extent to which estrogen-sensitive, ER-dependent uterine organizational events characteristic of the first 2 weeks of postnatal life may determine adult uterine and endometrial responses to the conditions of pregnancy is incompletely defined.

Objectives of the present study were to evaluate long-term effects of neonatal EV treatment (from PND 0 through PND 13) on uterine and endometrial responses to conditions characteristic of the periattachment period on Day 12 after estrus/mating in adult gilts. The hypothesis underlying these investigations was that uterine capacity to respond to pregnancy is altered by neonatal EV exposure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All animals were housed at the Auburn University Swine Research and Education Center. Animal procedures were approved by the Auburn University Institutional Animal Care and Use Committee and were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [16].

Experimental Design

The experimental design is depicted in Figure 1. On PND 0, 36 white crossbred gilts were assigned randomly to one of two neonatal treatment groups (n = 18 gilts/group). Care was taken to ensure that treatments were balanced for potential effects of litter. Treatment assignments were made randomly among gilts within a litter, and sows were nursing litters of similar size. Neonatal treatments, administered as daily injections from birth (PND 0) through PND 13, were either corn oil vehicle (CO; 50 µl kg body weight-1 day-1, i.m.) or EV (50 µg kg body weight-1 day-1, i.m.). Gilts were maintained together throughout the study. No further exogenous treatments were administered after PND 13. Also at birth, gilts were assigned randomly to either remain cyclic (CY) or to be bred at the second observed estrus. Thus, the experiment was designed to generate adult uterine tissues from CY and pregnant (PX) gilts on Day 12 after estrus/mating that were either EV exposed or unexposed (CO) from PND 0 to PND 13.



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  		FIG. 1. Experimental design. At birth (PND 0), crossbred gilts (n = 36) were assigned to one of two treatment groups (n = 18 gilts/group) and received daily injections of either CO (50 µl kg body weight-1 day-1) or EV (50 µg kg body weight-1 day-1) from PND 0 through PND 13. Estrus detection began on approximately PND 170. Nine gilts from each treatment group were assigned to remain cyclic (CY) or be bred (PX) at the second postpubertal estrus. All gilts were hysterectomized on Day 12 after estrus/mating

Beginning on approximately PND 170, gilts were observed once daily (at 0630 h) for estrus. At the second estrus (designated Estrous Cycle Day 0), nine gilts from each neonatal treatment group (CO and EV) were bred by natural service. Gilts assigned to be bred were serviced at first observation of estrus and again 24 h later. Together, these gilts comprised the CO/PX and EV/PX groups. The remaining 18 gilts (CO/CY and EV/CY) were not bred and served as sources of endometrial tissues from Day 12 of the estrous cycle.

Tissue Collection, Processing, and RNA Isolation

Gilts were sedated by i.m. injection of telazol (2.2 mg/kg body weight) and xylazine (2.2 mg/kg body weight). Uterine tissues were obtained by hysterectomy of gilts maintained with halothane in a surgical plane of anesthesia. All tissues were kept on ice throughout processing. Immediately after hysterectomy, uteri were trimmed free of cervix, oviducts, ovaries, and supportive connective tissue, rinsed of any blood, and briefly blotted dry with absorbent paper. The number of corpora lutea on each ovary was recorded.

Uteri were weighed, and the length and diameter of each uterine horn were determined as described previously [15]. These measurements were recorded, and data for uterine horn length and diameter were used later to estimate uterine horn volume using the equation for the volume of a cylinder [15].

Uterine horns were flushed individually with 20 ml of cold sterile saline (0.9% NaCl, w/v) as described previously [15] to collect resident uterine luminal fluid (ULF) from CY and PX gilts and conceptuses from PX gilts. ULF was collected into sterile 100- x 90-mm Petri dishes and examined for the presence and relative state of development of conceptuses. Conceptuses were discarded, and individual ULF samples were frozen and stored at -80°C until assayed for total protein using a protein assay (Bio-Rad, Hercules, CA). Prior to protein assays, all ULF samples were clarified by centrifugation.

After collection of ULF, each uterine horn was opened along its mesometrial border and endometrium was harvested using a scalpel and forceps. Histologic evaluation of endometrium collected in this way revealed no evidence of significant contamination by myometrium. Harvested endometrium was either processed for explant culture or frozen immediately in liquid nitrogen and stored at -80°C. RNA was isolated from frozen tissues using Trizol reagent as directed by the manufacturer (Invitrogen, Carlsbad, CA). Each RNA sample was evaluated spectrophotometrically, and integrity of ethidium bromide-stained RNA was assessed following electrophoresis through 1.5% agarose gels [17].

Endometrial Explant Culture

Endometrium, collected as described above, was placed immediately into a sterile tube containing phenol red-free culture medium consisting of Dulbecco modified Eagle medium (DMEM) nutrient mixture F-12 Ham base (DMEM/F12). Content of nonradioactive L-leucine in DMEM/F12 was limited to 5.2 µg/ml [15]. Duplicate cultures were established for each gilt as described previously [15].

The ability of explanted tissues to incorporate 3H-leucine (3H-Leu) into nondialyzable macromolecules likely to be representative of proteins synthesized and secreted by the endometrium was determined following extensive dialysis of individual samples in Spectra-Por 7 dialysis tubing (MWCO = 1000) against 10 mM Tris-HCl (pH 8.2) [5, 15]. The amount of 3H-Leu present in pre- and postdialysis aliquots of culture medium was determined by liquid scintillation spectrometry. These data were used to calculate the percentage incorporation of 3H-Leu into nondialyzable endometrial products for each culture.

Radioimmunoassay

Concentrations of estradiol in ULF were determined by RIA, using a Coat-A-Count kit (Diagnostic Products Corporation, Los Angeles, CA), following the manufacturer's directions. For each gilt, ULF estradiol for each uterine horn (left and right) was assayed in duplicate 200-µl samples. Validation assays included quantitative detection of added estradiol to ULF and confirmation of parallelism, over the range of 20–500 pg/ml, between diluted ULF samples and the standard curve, which was prepared in 0.9% NaCl. Assay sensitivity was 20 pg/ml. All samples were processed in a single assay. The intra-assay coefficient of variation was 5%. Total estradiol recovered per gilt was calculated using the data for recovered ULF volume. For each gilt, total estradiol content per uterine flush (left and right horns) was determined by multiplying estradiol concentration by flush volume recovered. Total estradiol content per gilt was then estimated by adding values for left and right ULF samples.

RNase Protection Assays

Effects of neonatal estrogen-induced disruption of uterine development on levels of mRNA present in endometrium obtained from adult gilts on Day 12 of the estrous cycle (CY gilts) or pregnancy (PX gilts) were evaluated by RNase protection assay (RPA). Assays were developed previously for ER, progesterone receptor (PR), uteroferrin (UF), and retinol-binding protein (RBP) with porcine endometrial RNA [15]. An RPA was optimized to determine levels of mRNA for keratinocyte growth factor (KGF). All of these mRNAs were chosen as markers of steroid-sensitive endometrial function at the transcriptional level [18–24].

All RPAs were conducted using the RPA III kit (Ambion, Austin, TX). For all assays, an antisense cyclophilin cRNA probe transcribed from the pTRI-cyclophilin human template (Ambion) was included at constant molarity in each sample to monitor RNA loading and variation due to processing [15].

Radiolabeled antisense cRNA probes for ER, PR, UF, and RBP were generated as previously described [15] using a MaxiScript in vitro transcription kit (Ambion) to incorporate [{alpha}-32P]uridine 5'-triphosphate (800 Ci/mmol; ICN Pharmaceuticals, Costa Mesa, CA). An antisense KGF probe was generated from the same template used by Ka and coworkers [24]. All probes were gel purified prior to use in RPAs.

Pilot assays were conducted to optimize RPAs using a pool of total endometrial RNA from CY and PX gilts following procedures described earlier [15]. Pilot assays were performed to determine the optimum amount of total endometrial RNA for each RPA and to identify the appropriate amount of diluted RNase A and RNase T1 mix for each target mRNA. The amount of total endometrial RNA assayed for each transcript was 10 µg.

Statistical Analyses

Data were subjected to least-squares ANOVAs using the general linear models procedures of SAS [25]. Error terms used in tests of significance were identified based upon the expectations of the error mean square [26]. All quantitative data are presented as least-squares means with SEMs. For uterine morphologic responses, ULF protein content, and percentage incorporation of 3H-Leu into nondialyzable endometrial products in vitro, statistical models considered variation due to treatment (CO vs. EV), pregnancy status (CY vs. PX), and the interactions of these terms.

For RPA data, linear relationships were identified for both target mRNA and cyclophilin signals over an appropriate range of potential loading values between 2.5 and 25 µg of total RNA. Preliminary analyses were conducted to confirm that levels of endometrial cyclophilin mRNA were independent of neonatal EV treatment. Because levels were not affected by EV treatment, data for cyclophilin mRNA levels were used as a covariate to adjust for procedural variations in analyses. Data for levels of KGF mRNA were transformed to log10 prior to analysis to ensure homogeneity of error variance. Each assay was conducted within two gels. A reference sample of pooled RNA was assayed in duplicate on each gel to monitor intergel assay variability. Thus, overall statistical models for RPA data considered variation due to treatment, pregnancy status, treatment x pregnancy interaction, and gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Luteal and Uterine Morphometric Data

Neonatal treatment did not affect number of corpora lutea in adult gilts on Day 12 after estrus/mating. Mean number of corpora lutea per gilt was 15.5 ± 0.5.

Data for uterine weight and uterine horn volume are presented in Figure 2. Overall, neonatal EV exposure decreased uterine weight (Fig. 2A; Treatment, P < 0.001) and uterine horn volume (Fig. 2B; Treatment, P < 0.01) in adult gilts on Day 12 after estrus/mating. Both of these responses increased (P < 0.001) in PX compared with CY gilts. However, a treatment x status interaction (P < 0.01) for these responses indicated that the pregnancy-associated 40% increase in uterine weight (Fig. 2A) and 48% increase in uterine horn volume (Fig. 2B) observed in CO gilts did not occur in gilts exposed neonatally to EV.



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  		FIG. 2. Effects of treatment with EV, administered from PND 0 to PND 13, on uterine wet weight (A) and uterine horn volume (B). Uterine tissues were collected on Day 12 after estrus/mating from CY and PX gilts. Significant treatment x status interactions (P < 0.01) were detected for both responses

ULF Estradiol and Protein Content

No effect of treatment was detected for ULF estradiol content. However, as expected, an effect of pregnancy status (P < 0.001) was observed. Estradiol was undetectable in ULF from CY gilts, whereas mean ULF estradiol content in PX gilts was 44.4 ± 7.0 ng.

When considering overall means for CY and PX gilts, neonatal EV reduced total protein content (Fig. 3; Treatment, P < 0.01) in ULF obtained on Day 12 after estrus/mating (CO, 23.6 mg; EV, 16.6 mg; SEM = 0.62 mg). Pregnancy increased total protein content (Status, P < 0.001) of ULF on Day 12 postmating (CY, 14.2 mg; PX, 26.0 mg; SEM = 0.62 mg). However, a treatment x status interaction (P < 0.01) was detected for this response. Although ULF protein content was greater in PX than in CY gilts from both treatment groups, the magnitude of this pregnancy-associated increase was markedly greater in CO (130%) than in neonatally EV-exposed gilts (36%).



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  		FIG. 3. Effects of treatment with EV, administered from PND 0 to PND 13, on ULF protein content. Uterine tissues were collected on Day 12 after estrus/mating from cyclic and pregnant gilts. A treatment x status interaction (P < 0.01) was detected for this response

Endometrial Incorporation of 3H-Leu into Nondialyzable Products In Vitro

For all gilts, neonatal EV reduced in vitro incorporation of 3H-Leu into nondialyzable products by endometrial tissues from gilts at Day 12 after estrus/mating (Treatment, P < 0.05; CO, 14.9%; EV, 12.5%; SEM = 0.3%). No effects of pregnancy status were detected for this response (CY, 14.0%; PX, 13.4%; SEM = 0.3%).

Endometrial Transcript Levels

Data for relative levels of endometrial mRNAs in adult gilts on Day 12 after estrus/mating are presented in Figure 4. No effects of neonatal EV treatment were detected for levels of ER mRNA. However, ER mRNA levels were 54% lower in PX than in CY gilts (Status, P < 0.001). Neither treatment nor pregnancy status affected endometrial PR mRNA levels.



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  		FIG. 4. Effects of treatment with EV, administered from PND 0 to PND 13, on endometrial levels of mRNA for ER, PR, UF, RBP, and KGF, as determined by RPA. Uterine tissues were collected on Day 12 after estrus/mating from CY and PX gilts. Treatment (Trt), pregnancy status (Status), and treatment x status interactions were detected as indicated

No overall effect of neonatal treatment was detected for endometrial UF mRNA levels. However, levels of UF mRNA were 138% higher in PX than in CY gilts (Status, P < 0.001). Further, a treatment x status interaction (P < 0.01) indicated that the magnitude of this pregnancy-related response was attenuated in EV-exposed gilts. Effects of both treatment (CO < EV; Treatment, P < 0.10) and pregnancy status (CY < PX; Status, P < 0.07) were detected for endometrial levels of RBP mRNA. Overall, relative levels of endometrial RBP mRNA were higher in EV than in CO gilts and in PX than in CY gilts. In contrast, neonatal EV reduced endometrial KGF mRNA levels in both CY and PX gilts on Day 12 after estrus/mating (Treatment, P < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, EV was used to disrupt estrogen-sensitive, ER-dependent organizational events that support uterine development between birth and PND 13. In rodents, perinatal exposure to estrogen or related xenobiotics induces reproductive tract lesions [27]. Long-term effects of such exposures include genital tract hypoplasia, altered steroid responsiveness, and aberrant uterine function in adults [27]. Previous studies revealed that transient EV exposure from birth, as described here, resulted in uterine hypoplasia and altered endometrial steroid responses in prepubertal gilts [15] and decreased embryo survival in adults by 22% on Day 45 of gestation [1]. Present results extend these observations and indicate that the pig can be added to the list of species for which perinatal exposure to estrogen has long-term consequences for both uterine morphology and function in adults.

As reported for rodents [27–29], documented for the prepubertal pig [7, 15], and confirmed here in adult gilts, estrogen administered early in postnatal life is acutely uterotrophic but ultimately antiuterotrophic. Decreased uterine weight and size, similar to that observed for adult EV-exposed gilts, was also observed in adult ewes exposed to 19-norprogestin from birth and in adult beef heifers exposed from birth to a combination of estrogen plus progesterone [9–11, 30]. These data indicate that adult uterine size is programmed to some extent by steroid-sensitive organizational events characteristic of the early postnatal period. Mechanisms through which transient perinatal exposure to steroids or related xenobiotics may result in adult uterine hypoplasia are incompletely defined. However, such long-term organizational effects have been postulated to reflect the direct actions of developmentally disruptive steroidal agents on 1) expression patterns of genes, including members of both Wnt and abdominal-B Hoxa gene families [31], that determine or stabilize critical morphogenetic and cytodifferentiative events, 2) recruitment of cells into and their progression through the cell cycle, and 3) steroid withdrawal-induced apoptosis leading to reproductive tract hypoplasia [15]. Whatever the mechanism, developmentally induced uterine hypoplasia is typically associated with reduced uterine capacity for conceptus support.

A primary objective of this study was to determine whether transient neonatal EV exposure from birth would affect uterine responses to endocrine and conceptus signals in adult gilts on Day 12 after estrus/mating. In CY gilts, the absence of conceptuses in the uterine lumen at this time ensures luteolysis and continuation of cyclicity [32]. In PX gilts, the presence of conceptuses in the uterine lumen at this time and appropriate uterine integration of maternal endocrine and local conceptus signals ensure maternal recognition and maintenance of pregnancy [32, 33]. On Days 11–12 of gestation, porcine blastocysts expand rapidly, from spherical to filamentous, and begin to secrete significant amounts of estrogen into the uterine lumen [34, 35]. Coincident with these changes are uterine growth and synchronized endometrial secretion of proteins (histotroph) into the uterine lumen [36, 37]. Molecular markers of endometrial responsiveness to endocrine and conceptus signals at this stage of the periattachment period also include upregulated expression of UF [38], RBP [30, 39, 40], and KGF [23, 24]. These acute uterine responses to periattachment conceptus signals are considered essential for conceptus survival and reproductive success.

As expected [1], neonatal EV treatment at PND 0–13 did not affect ovulation rate or prevent conception in adult gilts. In every case, tubular and filamentous conceptuses were found in ULF obtained from PX gilts on Day 12 postmating. The fact that neither ovulation rate nor ULF estradiol content differed between PX CO and EV gilts suggests that the number of viable estrogen-active periattachment conceptuses contributing to the ULF estrogen pool was similar for these two groups of pregnant animals [41]. Therefore, to the extent that intrauterine conceptus signal intensity was similar between CO and EV gilts, data indicate that EV-induced disruption of uterine organizational events in the neonate altered the capacity of adult tissues to respond normally to conditions of pregnancy characteristic of the periattachment period at several levels.

The uterine growth response to early pregnancy, observed in CO gilts and reported by others [42], was absent in EV-treated PX gilts. The absence of this relatively acute uterine growth response to pregnancy indicates neonatally EV-induced dysregulation of mechanisms in the adult uterus that would normally regulate short-term effects of conceptus estrogen on events including water imbibition and tissue hydration [43]. Data for ULF protein content support this concept.

In contrast to the protracted release of endometrial secretory products seen during diestrus in normal CY gilts, the porcine endometrium of pregnancy mounts a dramatic, acute, and synchronized release of glandular secretory products in response to conceptus-produced estrogen [36, 44, 45]. This response, observed in CO/PX gilts as a 130% increase in ULF protein content over that observed for CO/CY gilts, was markedly attenuated in EV/PX gilts, in which the increase in ULF protein content over that observed for EV/CY gilts was only 36%. Reduced uterine secretory responsiveness in EV/PX gilts may reflect reduced endometrial capacity for protein synthesis, as indicated by reduced incorporation of 3H-Leu into nondialyzable products by tissues obtained from neonatally EV-exposed gilts. Data can also be interpreted to indicate that the course of estrogen-sensitive uterine developmental events characteristic of the neonatal exposure period targeted here ultimately dictates the extent to which mechanisms responsible for conceptus-induced, calcium-dependent endometrial secretory responsiveness to periattachment conceptus signals [45] become organized to function optimally. Unequivocal evidence of the essential nature of endometrial secretory competence for conceptus survival, growth, and maintenance of pregnancy was recently demonstrated in sheep [13, 46] and suggested earlier for the pig [37]. In the pig, combined effects of reduced uterine protein biosynthetic capacity and endometrial secretory competence, detectable by Day 12 of gestation in EV/PX gilts, are likely to represent major factors responsible for reduced embryo survival observed in neonatally EV-exposed gilts by Gestational Day 45 [1].

Endometrial ER and PR expression is required for tissue responsiveness to cognate ligands. Estrogen can autoregulate ER expression [19, 47], and PR is a classic estrogen target gene [20]. It was, therefore, important to determine whether relative levels of ER or PR mRNA were affected by neonatal EV exposure in the adult endometrium. To the extent that mRNA expression can be related to expression of ER [6] and PR proteins, the present data indicate that both receptor populations were present at similar levels in endometrial tissues obtained from all adult gilts and, therefore, that these tissues were capable of recognizing, if not responding optimally to, relevant steroid hormone signals. Thus, the neonatally EV-induced uterine lesion described here was not documented at this level. In contrast to an earlier report [48], in which ER mRNA levels were quantified by dot blot analysis, reduced levels of endometrial ER mRNA were detected here for PX as compared with CY gilts on Day 12 after estrus/mating using the more sensitive RPA technique. This endometrial response to pregnancy may reflect local effects of conceptus estrogens present in ULF.

Although not observed for ER or PR, neonatal treatment effects at the level of the transcriptome were observed for three molecular markers of adult porcine endometrial function: UF, RBP, and KGF. As reported for prepubertal gilts [15] and rodents exposed to estrogens developmentally [49–51], responses at this level were transcript specific.

Synthesis and secretion of UF, a progesterone-inducible iron transport molecule and major uterine-derived protein component of porcine ULF, are regulated during early pregnancy by conceptus estrogen [36, 38, 45, 52]. Although constitutive levels of endometrial UF mRNA were higher for EV/CY than for CO/CY gilts, the pregnancy-associated increase in mRNA levels expected for UF and observed in CO/PX gilts was attenuated in EV/PX gilts in a manner similar to that observed for ULF protein content in EV/PX gilts. In contrast, constitutive expression of RBP mRNA, the translational product of which is also found in ULF [40], was generally upregulated by neonatal EV irrespective of pregnancy status. Levels of KGF mRNA, the translational product of which is expressed by endometrial epithelium, upregulated on Day 12 of pregnancy, and thought to stimulate growth of the trophectoderm [23, 24], were generally downregulated in both CY and PX neonatally EV-exposed adult gilts. Significant differential treatment effects observed for this targeted set of endometrial transcripts indicates that developmental disruption of critical uterine organizational events during early postnatal life can affect adult uterine phenotype at the level of the transcriptome. The mechanisms, extent, and consequences of such effects remain to be defined. However, mechanisms regulating both constitutive and pregnancy-induced endometrial expression of specific genes can be altered epigenetically and are programmed, to some extent, developmentally.

Overall, results of this and previous studies [1, 5, 7, 15] indicate that estrogen-sensitive developmental events characteristic of early postnatal life in the pig include elements of the organizational program governing morphogenetic and cytodifferentiative processes that determine the functional capacity of adult uterine tissues to accommodate pregnancy. Data also establish the neonatally estrogen-exposed porcine uterus as a useful and relevant model system with which to identify cellular and molecular elements of the uterine developmental program that ultimately dictate uterine capacity and reproductive efficiency in adults.


   ACKNOWLEDGMENTS
 
The authors thank Dr. W. Frank Owsley, Mr. Michael W. Carroll, Mr. Brian Anderson, Mr. Clinton Dowdell, and Mr. Ben Brown of the Auburn University Swine Research and Education Center for oversight and assistance with swine husbandry and breeding; Ms. Mabel H. Robinson for laboratory support; Mr. Jon Chervenak, Ms. Katherine Fontanetta, Mr. Andrew Simmons, and Mr. Seth Taylor for their help with animal-related activities and tissue processing; and Ms. Sarah McLean for tissue processing assistance.


   FOOTNOTES
 
1 This work was supported by USDA-NRI grant 98-3203-6198 to F.F.B. Back

2 Correspondence. FAX: 334 844 1519; bartoff{at}auburn.edu Back

3 Current address: Department of Obstetrics and Gynecology, Yale University, 333 Cedar Street, New Haven, CT 06510 Back

Received: 20 September 2002.

First decision: 8 October 2002.

Accepted: 30 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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