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Endocrinology, doi:10.1210/en.2004-0178
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Endocrinology Vol. 145, No. 7 3247-3257
Copyright © 2004 by The Endocrine Society

Neonatal Estrogen Exposure Disrupts Uterine Development in the Postnatal Sheep

Kanako Hayashi, Karen D. Carpenter and Thomas E. Spencer

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

Address all correspondence and requests for reprints to: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471. E-mail: tspencer{at}tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Postnatal development of the ovine uterus between birth and postnatal day (PND) 56 involves budding differentiation of the endometrial glandular epithelium from the luminal epithelium (LE) followed by extensive coiling and branching morphogenesis of the tubular glands. To determine the short- and long-term effects of estrogen on neonatal ovine uterine development after PND 14, neonatal sheep were randomly assigned at birth (PND 0) to be treated daily with estradiol-17ß benzoate (EB; 0, 0.01, 0.1, 1, or 10 µg/kg body weight·d) during one of two developmental periods (PND 14–27 or 42–55). All ewes were hemiovariohysterectomized at the end of EB treatment on either PND 28 or 56, and the remaining uterine horn and ovary removed on PND 112. Immediate responses to EB treatment included dose- and age-dependent increases in uterine wet weight, thickness of the endometrium, myometrium, and LE, but decreases in endometrial glands on PND 28 and 56. Transient exposure to EB decreased gland number and thickness of the endometrium and LE on PND 112 but did not affect extrauterine reproductive tract structures. The mechanism of estrogen inhibition of uterine development did not involve effects on cell proliferation. Real-time PCR analyses found that EB exposure disrupted normal patterns of growth factor (IGF-I, IGF-II, fibroblast growth factor-7, fibroblast growth factor-10, and hepatocyte growth factor) and receptor mRNA expression in the uterus. Transient exposure of the neonatal ewe to estrogens during critical periods specifically alters growth factor networks that perturb normal development of the uterus, leading to permanent alterations in uterine structure and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH HISTOGENESIS OF the uterus is initiated in the fetus, uterine development is not completed until after birth in humans, domestic animals, and laboratory rodents (1, 2). The major developmental event in the postnatal uterus is the differentiation and development of the endometrial glands. Uterine gland development is a critical event, because alteration or ablation of endometrial glands and/or their secretory products compromises survival and growth of the conceptus (embryo/fetus and associated extraembryonic membranes) in the mouse, rat, pig, cow, and sheep (1, 3, 4, 5). In humans, the secretory products of endometrial glands are also an important source of nutrition for conceptus growth during the first trimester (6). Consequently, the success of neonatal uterine development determines, in part, the embryotrophic and functional capacity of the uterus in the adult.

In sheep, uterine development after birth involves differentiation of the endometrial glandular epithelium (GE) from the luminal epithelium (LE), specification and development of the intercaruncular endometrial stroma, development of endometrial folds, and, to a lesser extent, growth of endometrial caruncular areas and the myometrium (7, 8, 9). Endometrial gland differentiation and development, which is also termed adenogenesis, in the sheep begins between postnatal days (PND) 1 and 7, when shallow epithelial invaginations appear along the LE in presumptive intercaruncular areas. Between PND 7 and 14, the nascent GE buds proliferate into the stroma and form tubules or ducts that begin to coil and branch at the tips by PND 21. After PND 21, uterine adenogenesis primarily involves coiling and branching morphogenesis of tubular endometrial glands as they proliferate through the lower stroma (e.g. stratum spongiosum) to the inner circular layer of the myometrium. By PND 56, uterine morphogenesis is essentially complete, because the aglandular caruncular and glandular intercaruncular endometrial areas appear histoarchitecturally similar to that of the adult uterus (9). Uterine adenogenesis is a critical event in sheep, because inappropriate exposure to progestins from birth to only PND 56 permanently ablates endometrial gland development and results in a uterine gland knockout phenotype in the adult (10). Adult uterine gland knockout ewes are infertile and exhibit a defect in periimplantation conceptus survival and growth (2, 4).

In the neonatal sheep, pituitary prolactin (PRL) and uterine stromal growth factors, including fibroblast growth factor (FGF)-7 and FGF-10, hepatocyte growth factor (HGF), and IGF-I and IGF-II, with their respective epithelial receptors [FGF receptor (FGFR)2 or FGFR2IIIb, c-met, and type I IGF receptor], have been implicated as endocrine and paracrine regulatory systems controlling postnatal endometrial adenogenesis (8, 9, 11, 12, 13). The IGF system is also implicated in postnatal rodent uterine development (14, 15). Expression of both short and long forms of the PRL receptor (PRLR) is restricted to the nascent GE buds on PND 7 and proliferating and developing GE from PND 14–56 (9). Recent evidence strongly supports a primary regulatory role for pituitary PRL in endometrial gland growth and branching morphogenesis in the neonatal ovine uterus (12). After PND 14, the ovary also influences uterine growth and endometrial gland morphogenesis, which may involve the activin-follistatin system (13, 16).

The role of estrogen and estrogen receptor {alpha} (ER{alpha}) in neonatal uterine development is species-specific. Postnatal uterine development is accompanied by expression of ER{alpha} in both the nascent and developing glands and endometrial stroma in rodents, pigs, and sheep (9, 17, 18, 19, 20, 21). Studies in rodents indicate that endometrial adenogenesis is not dependent on the ovary, adrenal gland, or uterine ER{alpha} (22, 23, 24, 25). Although initial neonatal uterine growth and endometrial adenogenesis in the pig are also ovary- and estrogen-independent (21), expression and activation of ER{alpha} is required, because treatment of neonatal gilts with ICI 182,870, an ER{alpha} antagonist, from birth inhibited endometrial gland development (26). In the neonatal ewe, the ovary does not produce appreciable amounts of estrogen, because the circulating levels of estradiol-17ß are negligible between birth and PND 56 (27). In contrast to the pig, treatment of ewes from birth with EM-800, an ER{alpha} antagonist, had no effect on uterine gland development on PND 14 and only moderately reduced the number of coiled and branched glands in the endometrium on PND 56 (27). The observed differences in effects of inappropriate exposure to estrogen on uteri of sheep and rodents, as compared with pigs, may be due to differences in effects of ovarian steroids on steroid receptor gene expression in endometrial epithelia and myometrium of the adult uterus. Transient disruption of the normal developmental program by exposure to estrogens, progestins, or related xenobiotics has long-term consequences for uterine function and reproductive health in humans, wildlife, laboratory animals, and domestic animals (1, 10, 28, 29, 30). Thus, studies are necessary to ascertain the cellular and molecular mechanisms mediating the developmental disturbances caused by exposure to endocrine disruptors, which could provide markers of exposure in the neonate and adult as well as therapies to counteract infertility in the adult.

Exposure of postnatal animals to estrogen or other ER agonists during critical developmental periods in the neonate induces a uterotrophic response and either inhibits or potentiates adenogenesis in the rat and pig, respectively (26, 31, 32, 33). In sheep, exposure of the developing uterus to estrogen from birth reduced uterine growth and completely ablated endometrial adenogenesis as assessed on both PND 14 and 56 (27). Our working hypothesis is that exposure of neonatal sheep to estrogens during the tubulogenic and coiling and branching morphogenetic periods of uterine development may positively affect uterine glands. Therefore, the objective of this study was to determine the short- and long-term effects of transiently exposing neonatal sheep to estrogen on uterine development during two critical neonatal periods (PND 14–27 or 42–56) during the coiling and branching morphogenesis of uterine glands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
All experiments and surgical procedures were in accordance with the Guide for the Care and Use of Agriculture Animals in Agricultural Research and Teaching and approved by the University Laboratory Animal Care Committee of Texas A&M University. Crossbred Suffolk ewes were mated to Suffolk rams between September and November of 2002. Pregnant ewes were maintained according to normal animal husbandry practices. Ewes included in the following experiment were born between January and May of 2003. At birth (PND 0), ewes (n = 50) were assigned randomly to receive daily im injections of estradiol-17ß benzoate (EB; Sigma Chemical Co., St. Louis, MO) dissolved in corn oil vehicle at a dose of either 0, 0.01, 0.1, 1, or 10 µg/kg body weight (BW) from PND 14–27 (period one) or PND 42–55 (period two). Ewes were weighed, and the EB dose was adjusted every 7 d.

To determine the immediate effects of estrogen exposure on reproductive tract development, ewes were weighed and surgically hemiovariohysterectomized 24 h after the last treatment with EB on either PND 28 (period one) or PND 56 (period two). Briefly, the right ovarian pedicle was ligated with suture, and the right ovary was removed and weighed. The right uterine horn was ligated with suture immediately above the intercornual ligament, and the anterior portion of the right uterine horn above the ligature was removed. The oviduct was trimmed off, and then the right uterine horn portion was measured for length and weighed. Two pieces (~1 cm) of the middle region of the uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2) at room temperature for 24 h and processed for histology. The remainder of the uterine horn was frozen in liquid nitrogen and stored at –80 C for RNA extraction. On PND 112, all ewes were weighed, and the entire reproductive tract was removed. The left ovary was trimmed free of the mesovarium, weighed, and fixed in 4% paraformaldehyde in PBS fixative. The uterus was obtained and trimmed free of the broad ligament, oviduct, and cervix. The entire left uterine horn was dissected from the remaining portion of the right uterine horn, weighed, and measured for length and circumference. Sections (~1 cm) from the midportion of the uterine horn were fixed in 4% paraformaldehyde fixative, and the remainder of the uterus was frozen in liquid nitrogen and stored at –80 C. In addition, the ovary, oviduct, cervix, and vagina were fixed in 4% paraformaldehyde fixative. The left and right mammary glands were removed in their entirety and weighed.

PCR analysis
Total cellular RNA was isolated from frozen uterine tissue using Trizol reagent (Life Technologies, Inc.-BRL, Bethesda, MD) according to manufacturer’s recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively.

The cDNA was synthesized from uterine total RNA (5 µg) using random primers (Invitrogen, Carlsbad, CA), oligo(deoxythymidine) primers, and SuperScript II Reverse Transcriptase (Invitrogen) as described previously (34). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl water, and stored at –20 C. PCR analysis of mRNA expression was performed using an ABI PRISM 7700 (Applied Biosystems, Foster City, CA) with SYBR Green PCR Master Mix (Applied Biosystems) as the detector, according to manufacturer’s recommendations, as described previously (35). Primers were designed to amplify cDNAs of less than 100 bp to maximize efficiency (Table 1Go). PCR cycle parameters were 95 C for 15 sec and 60 C for 1 min for 40 cycles. Data were analyzed by using GeneAmp 5700 SDS software (version 1.4, Applied Biosystems). The threshold line was set in the linear region of the plots above the baseline noise, and threshold cycle (CT) values were determined as the cycle number at which the threshold line crosses the amplification curve. PCR without template or template substituted with total RNA was used as a negative control to verify experimental results. The results are expressed as fold increase or decrease in relative mRNA expression of the target gene. The fold changes are equivalent to 2(x – y), where x is the CT value of the control (0 µg EB), and y is the CT value of the treated ewes (1 µg or 10 µg EB dose).


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  		TABLE 1. Sequences of primers for real-time RT-PCR

 
Histology and morphometry
After fixation in 4% paraformaldehyde in PBS, uterine tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast Plus (Oxford Labware, St. Louis, MO) at the Texas A&M University College of Veterinary Medicine Histology Laboratory. Uteri were sectioned (5 µm) and stained with hematoxylin and eosin (H&E) as described previously (36). Uterine sections (n = 4) from each ewe were photomicrographed, and images were analyzed using Scion Image software (Scion Corporation, Frederick, MD) as described previously (12). Measurements were standardized using the image of a stage micrometer at the same magnification. The number of superficial ductal invaginations of GE from LE into the stroma was determined. The criterion for a ductal gland invagination was an invagination of the GE into the underlying stroma with a length of at least 10–20 µm and could be visibly tracked to a cross-section of a gland. Endometrial gland number was determined by counting the total number of uterine glands in a complete cross-section of the uterine horn. A gland cross-section with a visible open lumen was counted as a single uterine gland. Endometrial gland density was determined by counting the number of glands in a 0.04-mm2 (PND 28 and 56) or 0.25-mm2 (PND 112) area of the stratum compactum and stratum spongiosum areas, respectively, of the intercaruncular endometrium. The number of ductal gland invaginations and endometrial gland number and density estimates were generated for at least three areas within five nonsequential sections from each uterine horn. Intra- and intersection repeatability estimates for determination of ductal gland invagination number and endometrial gland number by a single observer was 0.85 and 0.8, respectively. The thickness or width of the intercaruncular endometrium and myometrium (inner circular and outer longitudinal layers), as well as LE cell height, was measured using the Scion Image software from multiple points (n = 4) of at least 10 nonsequential uterine sections.

Immunohistochemistry
Immunoreactive proliferating cell nuclear antigen (PCNA) and ER{alpha} proteins were localized in cross-sections (5 µm) of the uterus using the appropriate mouse antibodies and a Super ABC Mouse/Rat IgG Kit (Biomeda, Foster City, CA), using methods described previously (9). Mouse monoclonal antibody to PCNA (M0879; clone PC10) was purchased from Dako (Carpinteria, CA). Rat monoclonal antibody to human ER{alpha} (H222) was kindly provided by Dr. Geoffrey Greene (University of Chicago, Chicago, IL). The final working antibody concentration was 1 µg/ml for PCNA and 4 µg/ml for ER{alpha}. Antigen retrieval using boiling citrate buffer was performed as described previously for PCNA detection (9, 11). Antigen retrieval using limited pronase digestion was performed as described previously for ER{alpha} detection (37). The chromagen used for peroxidase localization was 3,3'-diaminobenzidine tetrahydrochloride from Sigma Chemical Co. Negative controls were performed in which the primary antibody was substituted with the same concentration of purified normal mouse IgG from Sigma Chemical Co. Multiple tissue sections from each ewe were processed as sets within an experiment.

As described previously (8), relative hybridization signal intensity for staining intensity for immunoreactive protein expression (ER{alpha}) was assessed visually in uterine sections from each ewe, by two independent observers, and scored as follows: absent (–; i.e. no staining above IgG control), weak (+), moderate (++), or strong (+++). Intercaruncular endometrial tissues, including LE, stroma, and GE, and myometrium, were scored. The GE and stroma was separated into shallow (stratum compactum) and deep (stratum spongiosum).

Photomicroscopy
Representative photomicrographs of uterine tissues were taken using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera. Digital images were captured and assembled using Adobe Photoshop (Adobe Systems, Seattle, WA).

Statistical analyses
All quantitative data were subjected to least-squares ANOVA using the General Linear Models procedures of the Statistical Analysis System (Cary, NC). Organ weight data were analyzed using bodyweight as a covariate. Analysis of PCR data incorporated the cyclophilin values as a covariate in the statistical model to correct for differences in the amounts of reverse transcriptase cDNA analyzed for each uterus. In all analyses, error terms used in tests of significance were identified according to the expectation of the mean squares for error. Orthogonal contrasts were used to determine effects of EB dose. Data are presented as least-squares means of untransformed values with overall SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Short-term effects of estrogen exposure on the neonatal ovine uterus
Ovary.
The weight of the ovary on PND 28 or 56 was not affected (P > 0.10) by treatment with EB (Tables 2Go and 3Go). The ovaries from both control and EB-exposed ewes contained numerous small antral follicles, and the number of these follicles was not affected by EB treatment (data not shown).


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  		TABLE 2. Effects of estrogen exposure during period one (PND 14–27) on the uterus and ovary on PND 28

 

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  		TABLE 3. Effects of estrogen exposure during period two (PND 42–55) on the uterus and ovary on PND 56

 
Uterus.
Short-term effects of EB exposure on uterine development were determined by removing a portion of the right uterine horn on either PND 28 or 56. Uterine wet weight (milligrams per centimeter) was calculated by dividing the wet weight of the horn by its measured length. Uterine horn length was not affected by EB exposure. Treatment of ewes with EB affected uterine horn wet weight depending on the dose of EB and developmental period of exposure (Tables 2Go and 3Go). Exposure of ewes to EB during the first developmental period (PND 14–27) increased uterine wet weight on PND 28 but only in ewes receiving 1 µg EB compared with control ewes. In contrast, exposure of ewes to all doses of EB during the second developmental period (PND 42–55) increased uterine wet weight on PND 56 in a dose-dependent manner (quadratic, P < 0.02).

Uterine histoarchitecture.
As summarized in Tables 2Go and 3Go and illustrated in Fig. 1Go, exposure of ewes to EB affected development of the uterus in a dose- and period-dependent manner. Histologically, the columnar LE appeared hypertrophic in ewes receiving higher doses of EB. The thickness of the endometrium and myometrium appeared to be increased by EB. In contrast, the number of endometrial glands appeared to be fewer in EB-treated ewes, particularly in the lower stratum spongiosum endometrium adjacent to the inner circular layer of myometrium. In ewes receiving the higher doses of EB, the myometrium appeared to be increased in thickness and somewhat disorganized. To determine the precise effects of EB treatment on uterine histoarchitecture, morphometrical analyses of the uterus were conducted (see Tables 2Go and 3Go).



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  		FIG. 1. Representative photomicrographs of the uterus on PND 28 and 56 in ewes. Effects of treatment are described in the text and summarized in Tables 2Go and 3Go. Tissues were prepared and stained using H&E. M, myometrium; S, stroma. Bars, 500 µm at low magnification and 100 µm at high magnification.

 
Period 1 (PND 14–27).
Consistent with the hypertrophic appearance of the LE in the endometrium, the width of the endometrial LE was affected by EB exposure (Table 2Go). The lower doses of EB decreased width of the LE, whereas LE width was increased in ewes receiving the 1-µg and 10-µg EB doses. Similarly, thickness of the intercaruncular endometrium was decreased in ewes receiving 0.01 µg EB, whereas it was increased at higher doses of EB. Exposure of ewes to 10 µg EB did not affect thickness of the intercaruncular areas of the endometrium. However, the increase in myometrial thickness was maximal at the 10-µg EB dose.

Exposure to EB affected development of the endometrial glands on PND 28. The number of ductal gland invaginations from the LE was increased by 0.1 µg EB but not affected by any other dose of EB. Ewes treated with either 0.01 or 10 µg EB had a reduced number of endometrial glands in their uterus. An approximate 2-fold reduction in the total number of endometrial glands was observed in ewes receiving 10 µg EB. Overall, EB treatment increased gland density in the stratum compactum but concomitantly decreased endometrial gland density in the stratum spongiosum.

Period 2 (PND 42–55).
Consistent with histological observations (Fig. 1Go), exposure of neonatal ewes to EB increased width of the LE in the intercaruncular areas of the endometrium (Table 3Go). Maximal increases in endometrial LE width were observed at the 10-µg EB dose. The thickness of the endometrium and myometrium was also increased by exposure to EB in a dose-dependent manner. The effect of EB on intercaruncular endometrial thickness was maximal at the 1-µg dose. Further, the 10-µg EB dose did not increase thickness of the intercaruncular endometrium as effectively as the 1-µg dose of EB. In contrast, the increase in myometrial thickness was maximal at the 10-µg EB dose.

Exposure to EB during period 2 (PND 42–55) affected endometrial gland development on PND 56. The number of ductal gland invaginations from the LE was decreased by exposure to EB in a dose-dependent manner. However, EB exposure decreased the total number of endometrial glands only at the 1-µg and 10-µg doses. Ewes receiving 10 µg EB exhibited an almost 2-fold reduction in the total number of endometrial glands. Although exposure of ewes to 0.01 µg EB decreased gland density in the stratum compactum, gland density in the stratum spongiosum was increased. However, gland density in both the stratum compactum and stratum spongiosum endometrium was decreased at the 10-µg dose of EB.

Long-term effects of estrogen exposure of the reproductive tract
The long-term effects of EB exposure on neonatal ovine uterine development was determined by removing the female reproductive tract on PND 112 in ewes transiently exposed to EB during developmental period 1 (PND 14–27) or period 2 (PND 42–55). The uteri from these ewes contained an intact left horn. PND 112 was selected because it is before puberty, when ovarian steroid hormones during the estrous cycle would begin to influence uterine histoarchitecture.

Ovary.
On PND 112, weight of the ovary was not affected (P > 0.10) by EB, regardless of the developmental period of exposure. Indeed, the ovaries from all ewes contained numerous small antral follicles, and the number of these follicles was not affected by EB treatment (data not shown).

Oviduct, cervix, and vagina.
As illustrated in Fig. 2Go, treatment with EB did not affect histoarchitecture and, by inference, development of the oviduct, cervix, or posterior vagina.



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  		FIG. 2. Representative photomicrographs of the oviduct, cervix, and vagina on PND 112 in ewes. Transient exposure to EB did not affect histoarchitecture of these extrauterine reproductive tract tissues. Tissues were prepared and stained using H&E. Bars, 200 µm.

 
Uterus.
Treatment of ewes with EB affected uterine horn wet weight depending on the dose of EB and developmental period of exposure (Tables 4Go and 5Go). Although exposure of ewes to EB during period 1 (PND 14–27) increased uterine wet weight on PND 28, this effect was not observed on PND 112 (Table 4Go). In contrast, exposure of ewes to EB during period 2 (PND 42–55) decreased uterine wet weight on PND 112 but only at the 10-µg EB dose (Table 5Go). Length of the right uterine horn on PND 112 was not affected (P > 0.10) by EB treatment in both developmental periods.


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  		TABLE 4. Effects of estrogen exposure during period one (PND 14–27) on the uterus and ovary on PND 112

 

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  		TABLE 5. Effects of estrogen exposure during period two (PND 42–56) on uterus and ovary on PND 112

 
Uterine histoarchitecture.
As illustrated in Fig. 3Go, exposure of ewes to EB affected development of the uterus in a dose- and period-dependent manner. Histologically, the columnar LE of the intercaruncular endometrium appeared hypotrophic in ewes receiving the highest doses of EB. The thickness of the endometrium and myometrium appeared to be affected by EB. In addition, the number of endometrial glands appeared to be fewer in EB-treated ewes, particularly in the lower stratum spongiosum endometrium adjacent to the myometrium. In ewes receiving the higher doses of EB, the myometrium appeared to be disorganized. Effects of EB exposure on uterine histoarchitecture were determined using morphometric analyses (see Tables 4Go and 5Go).



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  		FIG. 3. Representative photomicrographs of the uterus on PND 112 in ewes. Effects of treatment are described in the text and summarized in Tables 4Go and 5Go. Tissues were prepared and stained using H&E. Bars, 500 µm at low magnification and 100 µm at high magnification.

 
Period 1 (PND 14–27).
Transient exposure of neonatal ewes to EB decreased width of the LE in the intercaruncular areas of the endometrium on PND 112 (Table 4Go); an effect that was most pronounced at the 10-µg EB dose. The thickness of the endometrium and myometrium was moderately decreased by exposure to 10 µg EB.

The number of ductal gland invaginations from the LE into the stratum compactum stroma was decreased by all doses of EB. However, the total number of endometrial glands was decreased only in ewes treated with 1 µg or 10 µg EB. Ewes receiving 10 µg EB had a 2-fold reduction in the total number of endometrial glands, and this effect was attributed to a decrease in stratum spongiosum endometrial gland density.

Period 2 (PND 42–55).
Transient exposure of neonatal ewes to EB decreased thickness of the LE in the intercaruncular areas of the endometrium on PND 112 (Table 5Go). The thickness of the intercaruncular areas of the endometrium was decreased by exposure to EB in a dose-dependent manner, and this effect of EB was most pronounced in ewes receiving the 1-µg dose. In contrast, the thickness of the myometrium was increased by EB, with the maximum increase elicited by the 0.1-µg EB dose.

The number of ductal gland invaginations from the LE into the stratum compactum stroma was only decreased by EB at the 0.1-µg dose. However, a differential effect on endometrial gland number was observed in EB ewes. Treatment with 0.1 µg EB increased gland number, whereas treatment with 1 µg or 10 µg EB decreased endometrial gland number. The uteri of ewes exposed to 10 µg EB contained 1.4-fold fewer endometrial glands compared with control ewes. This reduction in uterine gland number was primarily due to decreased gland density in the stratum spongiosum.

Exposure to EB does not affect uterine cell proliferation
To determine effects of EB exposure on uterine cell proliferation, expression of immunoreactive PCNA protein was determined using immunohistochemistry. PCNA is a highly conserved DNA polymerase accessory protein essential for DNA synthesis, expressed during late G1 and S phases of the cell cycle, and a marker of cell proliferation (38). Immunoreactive PCNA protein was detected in all uterine cell types on PND 28, 56, and 112, and transient exposure to EB did not affect patterns of PCNA protein expression (data not shown). PCNA protein abundance was greatest in the LE and GE of the intercaruncular endometrial areas.

Exposure to EB reduces ER{alpha} expression
In control ewes on either PND 28, 56, or 112, immunoreactive ER{alpha} protein was most abundant in the endometrial GE but was detected at lower abundance in all other uterine cell types (Fig. 4Go and Table 6Go). The short-term effects of EB exposure during either period 1 or period 2 was a decrease in the abundance of ER{alpha} protein in the uterus, particularly in the endometrial LE and GE. In contrast, there appeared to be no differences in ER{alpha} expression in the uterus on PND 112.



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  		FIG. 4. Representative photomicrographs depicting expression of ER{alpha} protein in the uterus of control and EB-treated ewes on PND 28, 56, and 112. Immunolocalization of ER{alpha} protein using rat-antihuman ER{alpha} monoclonal antibody. Nuclear staining was not observed when irrelevant rat IgG was substituted for primary antibodies. Bar, 100 µm.

 

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  		TABLE 6. Distribution and relative abundance of immunoreactive ER{alpha} protein in the neonatal uterus from EB-treated ewes1

 
Effects of estrogen exposure on PRL-R, IGF-I, IGF-II, IGF-IR, FGF-7, FGF-10, FGFR2IIIb, HGF, and c-met mRNA expression in the uterus
To determine effects of estrogen exposure on patterns of uterine gene expression, real-time PCR analyses were conducted on uteri from control (0 µg) and EB (1 µg or 10 µg)-exposed ewes that were collected on either PND 28, 56, or 112 (Table 7Go). The 1-µg and 10-µg doses of EB were chosen due to their disruptive effects on uterine gland development.


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  		TABLE 7. Real-time PCR analysis of mRNA levels in the neonatal ovine uterus

 
Period 1 (PND 14–27).
Short-term effects of exposure to 1 µg EB during period 1 were to increase expression of mRNAs for several growth factors (IGF-I, FGF-7, FGF-10) and growth factor receptors (FGFR2IIIb and c-met) on PND 28. However, the 10-µg dose of EB only increased expression of FGF-10 but did not affect expression of other genes.

The long-term effects of EB exposure during period 1 on uterine gene expression were dose dependent as assessed on PND 112. Transient exposure to 1 µg EB only increased expression of FGF-7 in the uterus on PND 112. In contrast, transient exposure to 10 µg EB decreased expression of growth factors (FGF-7, FGF-10) and growth factor receptors (PRLR, c-met).

Period 2 (PND 42–55).
Short-term effects of exposure to 1 µg EB during period 2 were to decrease expression of IGF-IR mRNA but increase expression of several growth factors (FGF-7, FGF-10) and growth factor receptors (FGFR2IIIb and c-met) on PND 56. Exposure to the 10-µg dose of EB decreased expression of IGF-I, HGF, and IGF-IR but increased expression of FGF-7, FGF-10, and FGFR2IIIb.

The long-term effects of EB exposure during period 2 on uterine gene expression was assessed on PND 112. Transient exposure to both 1-µg and 10-µg doses of EB decreased expression of growth factors (IGF-I, IGF-II, FGF-7, FGF-10, HGF) and growth factor receptors (PRL-R, IGF-IR, FGFR2IIIb, c-met) on PND 112.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, transient exposure of postnatal sheep to EB during both developmental periods (PND 14–27 or 42–55) was found to disrupt uterine development and, in particular, endometrial adenogenesis. The present study complements previous findings that exposure of neonatal ewes to estradiol-17ß valerate (EV) from birth (PND 0) to PND 13 decreased uterine growth and ablated endometrial gland development on PND 14 (27). Indeed, exposure of postnatal ewes to norgestomet, a potent 19-norprogestin, from birth also reduced uterine growth and ablated endometrial gland differentiation (10, 11). These results support the hypothesis that sheep enter a critical developmental period after birth that continues to at least PND 56, because exposure to steroid hormones and other substances during this period disrupts uterine development and function in the adult. Indeed, the uterus is sensitive to developmentally disruptive effects of estrogens and progestins during critical organizational periods in humans (39), rodents (31, 32, 40, 41, 42), cattle (43), and pigs (26, 33, 44, 45).

Inappropriate exposure of postnatal sheep to estrogen or progestins during the infantile period (PND 0–14) prevents budding differentiation of the GE from the LE in the endometrium (11, 27). In the present study, neonatal sheep were exposed to increasing doses of EB from either PND 14–27 or 42–55. During these two periods of uterine development, the tubular endometrial glands undergo coiling and branching morphogenesis as they proliferate in the stratum spongiosum stroma toward the inner circular layer of the myometrium (9). Overall, exposure of the developing ovine uterus to EB during period 1 (PND 14–27) did not produce a uterotrophic response but did decrease development of endometrial glands in the PND 28 uterus. In contrast, exposure of the developing ovine uterus to EB during period 2 (PND 42–55) elicited a pronounced increase in uterine wet weight as well as a decrease in endometrial gland number in the PND 56 uterus. The uterotrophic effects of EB were observed at all doses, whereas the antiadenogenic effects of EB were observed only at the high doses of EB (1 µg and 10 µg) during period 2. Therefore, the neonatal sheep uterus is more sensitive to the effects of EB as it matures from PND 14–56, which may reflect changes in histoarchitecture as well as competence to respond to hormones and complexity of intrinsic gene networks. Similarly, the uterotrophic and antiadenogenic effects of other ER agonists, such as E2, tamoxifen, and toremifene, are also dose and age dependent in postnatal rats (31, 32, 42).

In the present study, the short-term effects of EB exposure were to decrease endometrial gland development. This antiadenogenic effect was accompanied by induction of LE hypertrophy and increases in endometrial and myometrial thickness that were EB dose and developmental period dependent. In the postnatal rat, transient exposure to E2 or tamoxifen elicited a marked LE hypertrophy associated with cellular degeneration (31, 32). Histologically, cellular degeneration in the LE was not observed in association with LE hypertrophy. Proliferation of uterine cells, as detected by immunoreactive PCNA protein expression, in the postnatal sheep was not affected by treatment with EB or in similar studies in which neonatal ewes were exposed to estrogen or a progestin (11, 27). Collectively, these results support the hypothesis that the antiadenogenic effects of estrogens and progestins on the neonatal sheep uterus do not appear to result from apoptosis or degeneration of the endometrial LE that would ablate or reduce gland stem cells. However, the effects of estrogens and progestins during the exposure period on cell proliferation and degeneration have not been studied. In the present study, the short-term effects of exposure to EB were to increase thickness of the LE, endometrium, and myometrium. These changes in the uterine wall are likely to disrupt the nature of the extracellular matrix that mediates epithelial-stromal cell interactions and growth factor action that are required for uterine development (46).

After birth, all uterine cell types express ER{alpha} in the sheep uterus, with abundant expression observed in the nascent and proliferating endometrial glands (9). Thus, cell-type specific responses may be involved in the dose-related effects of EB on uterine development. In the present study, treatment of postnatal ewes with EB dose-dependently during either developmental period decreased ER{alpha} expression in the uterus, particularly in the endometrial glands. Similarly, treatment of ewes with either EV or norgestomet, from birth, ablated endometrial adenogenesis and suppressed ER{alpha} expression in the endometrial epithelia (11, 27). Although ER{alpha} does not appear to regulate budding differentiation of endometrial glands in the neonatal ovine uterus (9), ER{alpha} does regulate, in part, the coiling and branching morphogenetic development of endometrial glands between PND 14 and 56 (27). Therefore, an EB-dependent decrease in ER{alpha} expression in the epithelium may be responsible, in part, for the antiadenogenic effects of EB on the neonatal sheep uterus.

Available evidence supports the concept that EB effects on uterine development involve age-, dose-, and uterine cell type-dependent effects on intrinsic growth factor networks regulating epithelial-stromal interactions important for uterine wall morphogenesis (8). Previously, we determined that inappropriate exposure of the postnatal sheep to norgestomet and EV from birth differentially altered expression of components of the HGF, IGF, and FGF-7/-10 growth factor networks through effects on the ligand or receptor (11). Collectively, the present studies of uterine histoarchitecture and gene expression by PCR indicate that uterine development is complex, involving multiple growth factor networks, and is differentially sensitive to detrimental effects of estrogens. The complex, disruptive effects of estrogens and progestins on postnatal uterine development undoubtedly involve alterations in other genes and gene networks, such as the Hox and Wnt genes (47). Given the complexity of postnatal uterine development, a systematic functional genomics approach is needed to unravel the developmental biology of postnatal uterine development and complex effects of endocrine disruptors.

The long-term effect of perinatal estrogen or progestin exposure are antiuterotrophic in a number of species (10, 11, 32, 41, 44, 45, 48, 49). In the present study, exposure to EB during either period elicited long-term reductions in endometrial gland number. In the pig and rodent, transient exposure to estrogens also has permanent effects on uterine function in the adult (32, 44, 45). Exposure of gilts to EV from birth to PND 13 decreased uterine weight and horn volume, and uterine responsiveness to potentially embryotrophic signals (44, 45). Similarly, administration of tamoxifen, a mixed ER agonist/antagonist, to neonatal rats on PND 1–5 or 10–14 elicited a dose-related inhibition of uterine gland genesis that persisted to PND 26 or 60, respectively (32). In the present study, no detrimental effect of EB treatment was observed on the vagina, cervix, or oviduct in terms of histoarchitecture. These results are similar to findings in postnatal sheep exposed to norgestomet (10, 11). Future studies will be directed toward determining the effect of transient exposure of postnatal sheep to estrogen, during critical organization periods in neonatal life, on reproductive fitness and health in the adult.

In summary, these data indicate that transient exposure of postnatal sheep to estrogens during two critical developmental periods inhibits uterine gland ontogeny. These disruptive effects of estrogen are age- and dose-related and appear to be permanent. The antiadenogenic effects of EB were manifest in disruption of stromal-epithelial interactions due to changes in normal patterns of stromal growth factors and their receptors, which are expressed predominantly or exclusively in the uterine epithelium. Both estrogens (50) and antiestrogens (51, 52) administered to neonatal rats induce hypothalamic lesions, which, in turn, cause tonic ovarian estrogen secretion and persistent estrus. Because normal brain and ovary function is perturbed by neonatal estrogen exposure, it is difficult to determine the direct effects of estrogens on the uterus in the adult. Available evidence in sheep and pigs suggests that transient postnatal exposure to ovarian steroids, either estrogen or progesterone, does not impair development of the hypothalamic-pituitary axis or extrauterine reproductive tract structures (10, 45). Therefore, domestic animals may be very useful models to determine specific effects of exposure to endocrine disruptors, during neonatal life, on function of the uterus in the adult. These studies are important because transient exposure to estrogens, xenobiotics, and other endocrine disruptors has permanent and irreversible effects on uterine development and function in humans, wildlife, laboratory animals, and domestic animals that are manifest in decreased reproductive fitness and health in adult life (1, 10, 28, 29, 30).


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


   Footnotes
 
This work was supported by National Institutes of Health Grants HD38274 and P30 ES09106. K.H. is a Lalor Foundation Fellow.

Abbreviations: BW, Body weight; CT, threshold cycle; EB, estradiol-17ß benzoate; ER, estrogen receptor; EV, estradiol-17ß valerate; FGF, fibroblast growth factor; FGFR, FGF receptor; GE, glandular epithelium; H&E, hematoxylin and eosin; HGF, hepatocyte growth factor; LE, luminal epithelium; PCNA, proliferating cell nuclear antigen; PND, postnatal day(s); PRL, prolactin; PRLR, PRL receptor.

Received February 11, 2004.

Accepted for publication March 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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