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Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inducible protein 10), and IGLV (immunoglobulin V). These mRNAs were expressed predominantly in the endometrial epithelia (GSTM3, IDH1, PEC1, OAS1, and TEGT), stroma (IGFBP3), or immune cells (CXCL10 and IGLV). In study 2, effects of estrogen exposure on uterine gene expression were determined during three different critical developmental periods (PNDs 0–14, 14– 28, and 42–56). Estrogen exposure decreased expression of the SSH-identified genes, particularly those from PNDs 0–14. These studies suggest that estrogen disruption of postnatal uterine development involves period-specific effects on expression of genes predominantly in the endometrial epithelium. The SSH-identified, estrogen-disrupted genes represent new candidate regulators of postnatal endometrial adenogenesis.
developmental biology, estradiol, female reproductive tract, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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– 9]. Endometrial adenogenesis begins between Postnatal Days (PNDs) 1 and 7, when shallow epithelial invaginations appear along the LE in presumptive intercaruncular areas. Between PNDs 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, the tubular glands begin to coil and branch as they proliferate through the stroma 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 to be histoarchitecturally similar to those of the adult uterus [9]. Uterine adenogenesis is a critical developmental 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 (UGKO) phenotype in the adult [10]. Adult UGKO sheep exhibit recurrent early pregnancy loss because of endometrial insufficiency [2, 4].
In the neonatal sheep, anterior pituitary prolactin (PRL) and uterine endometrial stromal growth factors, including FGF7 (fibroblast growth factor 7) and FGF10, HGF (hepatocyte growth factor), and IGF1 (insulin-like growth factor 1) and IGF2, along with their respective epithelial receptors (PRLR [PRL receptor], FGFR2 [FGF-receptor 2IIIb], MET [HGF receptor or c-Met], or IGF1R [type I IGF receptor]) are implicated as endocrine and paracrine regulatory systems controlling postnatal endometrial adenogenesis [8, 9, 11–15]. Expression of both short and long forms of the PRLR is restricted to the nascent GE buds on PND 7 and proliferating and developing GE from PND 14 to 56 [9]. Recent evidence supports a primary regulatory role for 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, presumably through the activin-follistatin system, which is present in both the ovary and uterus [13, 16].
Exposure of neonates to estrogens during critical developmental periods induces a uterotrophic response and either inhibits or potentiates adenogenesis in the rat and pig, respectively [17–20]. In neonatal sheep, ESR1 (estrogen receptor 
) is expressed after birth in all endometrial cell types and is particularly abundant in the nascent and developing GE as well as in the surrounding stroma [8, 21]. Postnatal uterine growth and endometrial adenogenesis are estrogen-independent in sheep from birth to PND 56, although coiling and branching morphogenesis after PND 14 is, in part, dependent on activated ESR1 [21]. However, inappropriate exposure of neonatal sheep to 17ß-estradiol valerate (EV) from birth reduced uterine growth and completely ablated endometrial adenogenesis as assessed on both PND 14 and PND 56 [21], which is similar to findings in the UGKO phenotype that results from exposure of sheep from birth to PND 56 to a progestin [11]. Transient exposure of neonatal sheep to 17ß-estradiol benzoate (EB) during critical periods for tubulogenesis and coiling/branching morphogenesis in uterine development (PNDs 14–27 or 42–55, respectively), retarded endometrial adenogenesis and was associated with altered endometrial growth factor (FGF7, FGF10, HGF, IGF1, and IGF2) and growth factor receptor (FGFR2, IGF1R, MET, and PRLR) expression in a dose- and period-dependent manner [14, 15].
Postnatal uterine development and, specifically, endometrial adenogenesis is a complex process involving stromal-epithelial interactions that are carefully orchestrated by multifactorial gene networks [22]. Given that sheep exposed to EV from birth lack endometrial glands, comparison of their glandless endometrium to that of normal glandular sheep should be useful to identify genes regulating adenogenesis as well as overall uterine development. Therefore, objectives of the present study were 1) to determine the effects of exposure of neonatal sheep to EV during the initial tubulogenic stage of endometrial adenogenesis (PNDs 0–14) on candidate gene networks implicated in the regulation of postnatal uterine development and endometrial adenogenesis, 2) to identify and clone mRNAs responsive to antiadenogenic EV in the neonatal uterus using polymerase chain reaction (PCR)-based suppression subtraction hybridization (SSH), and 3) to determine the effects of inappropriate exposure of neonatal sheep to estrogens on expression of the SSH-identified mRNAs during three critical developmental periods.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 were approved by the University Laboratory Animal Care Committee of Texas A&M University. Sheep were obtained from the Physiology Field Laboratory of Texas A&M University. Ewes were maintained according to normal animal husbandry practices.
In all studies, full cross-sections (
1 cm) of the middle region of each 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.
Study 1 Cross-bred Suffolk ewes were mated to Suffolk rams between July and October of 2003. Ewes were born between October 2003 and January 2004. At birth (PND 0), ewes (n = 14) were assigned randomly to receive daily i.m. injections from PND 0 to PND 13 (period 1) of corn oil vehicle as a control (CX; n = 7) or EV (Sigma Chemical Co., St. Louis, MO) in corn oil at a dose of 50 µg/kg body weight (EV-50; n = 7). This dose of EV retards uterine growth and inhibits endometrial adenogenesis in the neonatal ewe [21]. Ewes were weighed on PND 7, and the EV dose was adjusted accordingly. On PND 14, all ewes were necropsied.
Study 2 Cross-bred Suffolk ewes were mated to Suffolk rams between September and November of 2002. Ewes included in the following experiments were born between January and May of 2003. As described previously [14], ewes (n = 20) were assigned randomly at birth (PND 0) to receive daily i.m. injections from PND 14 to PND 27 (period 2) or from PND 42 to PND 55 (period 3) of corn oil vehicle as CX (n = 5), or EB (Sigma Chemical Co.) in corn oil at a dose of 10 µg/kg (EB-10; n = 5). This dose of EB during the two exposure periods reduces uterine growth and retards endometrial adenogenesis in the ewe [14]. Ewes were weighed every 7 days, and the EB dose was adjusted accordingly. To determine the immediate effects of EB exposure on reproductive tract development, ewes were weighed and surgically ovariohysterectomized 24 h after the last treatment with EB at either PND 28 or PND 56.
RNA Isolation
Total cellular RNA was isolated from frozen uteri of each ewe using Trizol reagent (Gibco-BRL, Bethesda, MD) according to the manufacturer's recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively. For purification of polyadenylated mRNA, total RNA from uteri in study 1 were pooled within treatment, and polyadenylated mRNA was purified from 1 mg of pooled total RNA using the Oligotex mRNA Midi Kit (Qiagen, Valencia, CA).
Semiquantitative Reverse Transcription-PCR Analysis
Using semiquantitative reverse transcription (RT)-PCR methodology described previously [8, 13], expression of IGF1, IGF2, IGF1R, FGF7, FGF10, FGFR2 (IIIb splice variant), HGF, MET, PRLR, INHBA, ACVR1, ACVR2, and FST mRNAs was assessed in total RNA isolated from the uterus of CX and EV-treated ewes in study 1. Briefly, cDNA was synthesized from total cellular RNA (5 µg) isolated from neonatal uteri using random (Life Technologies, Gaithersburg, MD) and oligo-dT primers and SuperScript II Reverse Transcriptase (Life Technologies). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl of water, and stored at –20°C. The cDNAs were diluted (1:10–1:100) with water before use in PCR. Primers were designed to amplify partial cDNAs for ovine mRNAs as summarized in Table 1. The PCR reactions were performed using ExTaq DNA polymerase (TAKARA Bio, Inc., Otsu, Japan) and either Optimized Buffer D (Invitrogen, Carlsbad, CA) for IGF2, IGF1R, INHBA, and ACTB or Optimized Buffer F (Invitrogen) for IGF1, FGF7, FGF10, FGFR2, HGF, MSTR1, PRLR, FST, ACVR1, and ACVR2 according to manufacturer's recommendations. The amount of cDNA template, annealing temperature, and number of cycles used for PCR initially were optimized to ensure that final PCR conditions were within the linear range of amplification for each primer pair. The IGF1, IGF2, IGF1, FGFR2, and ACTB reactions had 1 µl of cDNA (1:10); FST, INHBA, ACVR1, and ACVR2 PCR reactions 2 µl of cDNA (1:10); FGF10 and HGF PCR reactions 3 µl of cDNA (1:10); FGF7 and PRLR PCR reactions 5 µl cDNA (1:10); and MET PCR reaction 5 µl of cDNA (1:100). All PCR reactions were performed at 95°C for 30 sec, 54–61°C for 1 min, and 72°C for 1 min. Exact annealing temperatures used for each primer pair are provided in Table 1. Cycle numbers were as follow: 25 for IGF2; 30 for FGF7, FGF10, FGFR2, HGF, MET, IGF1, IGF1R, FST, ACVR2, and ACTB; and 33 for PRLR, INHBA, and ACVR1. Following PCR, equal amounts of reaction product were analyzed using a 2% agarose gel, and PCR products were visualized by ethidium bromide staining. The amount of DNA present was quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using an AlphaImager (Alpha Innotech Corporation, San Leandro, CA), and data were expressed as relative light units. The ACTB (ß-actin) values were used as covariates in statistical analyses to correct for differences in the amounts of cDNA used for each sample.
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PCR-Selected SSH
The PCR-select cDNA subtraction kit (catalog no. 637401; BD Biosciences, San Jose, CA) was used to compare the two populations of mRNA from the uteri of CX and EV-treated ewes in study 1 and to obtain a library of differentially expressed cDNAs. The technique of PCR-selected SSH prevents undesirable amplification of common cDNAs [23]. Using the kit according to the manufacturer's recommendations, two types of PCR subtracted neonatal ovine uterine cDNA libraries were generated: a CX library with EV cDNAs subtracted (CX-EV) using CX cDNAs as tester and EV as driver, and an EV library with CX cDNAs subtracted (EV-CX) using EV cDNAs as tester and CX as driver. Subtracted cDNAs were cloned into pCR2.1 using the Invitrogen T/A cloning kit, and resulting libraries were propagated in UltraMAX DH5-FT competent cells.
Differential Screening of the CX Uterus Subtracted cDNA Library
Colonies (n = 400) from the CX-EV cDNA library were picked, and plasmid DNAs were prepared using the R.E.A.L. Prep 96 BioRobot Kit (Qiagen) according to the manufacturer's protocol. The purified plasmid DNAs were affixed to a Nytran Plus positively charged nylon membrane (Schleicher and Schuell, Keene, NH) using a slot-blot manifold (Schleicher and Schuell). Forward-subtracted (CX-EV) and reverse-subtracted (EV-CX) cDNA populations were prepared as described in the BD Biosciences PCR-Select Differential Screening Kit user manual and used as a probe in hybridization analyses of the slot-blot membranes. Briefly, cDNAs (25 ng each) were random prime-labeled using [-32P]CTP and a DECAprime II kit (Ambion, Inc., Austin, TX). The DNA slot blots were then probed using the radiolabeled CX or EV cDNAs and ExpressHybe (BD Biosciences). After hybridization and washing, radioactivity associated with slots was quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ). The differentially expressed cDNA clones were selected if the ratio of the CX-EV probe signal was threefold or greater than that of the EV-CX probe signal. The sizes of differentially expressed cDNA clones were determined by restriction enzyme digestion and agarose gel electrophoresis.
In Situ Hybridization
In situ hybridization analyses of uteri from all studies were conducted using methods described previously [24]. Briefly, deparaffinized, rehydrated, and deproteinated cross-sections (5 µm) of the uterine horns from each ewe were hybridized with radiolabeled sense or antisense cRNA probes generated from linearized plasmid DNA templates using in vitro transcription with [-35S]UTP. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Kodak, Rochester, NY), stored at 4°C for 2–28 days, and developed in Kodak D-19 developer. Slides were then counterstained with Gills modified hematoxylin (Stat Lab, Lewisville, TX), dehydrated through a graded series of alcohols to a xylene substitute, and protected with a coverslip. Images of representative fields of sections hybridized with antisense or sense cRNAs were recorded under bright-field or dark-field illumination with a Nikon Eclipse 1000 photomicroscope (Nikon Instruments, Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera using constant image acquisition parameters to ensure accurate comparison.
Slot-Blot Hybridization
Steady-state levels of mRNA in uteri of all ewes from each study were assessed by slot-blot hybridization as described previously [25]. Radiolabeled antisense cRNA probes were generated from linearized plasmid templates by in vitro transcription with [-32P]UTP. Denatured total endometrial RNA (10 µg) from each ewe was hybridized with radiolabeled cRNA probes. To correct for variation in total RNA loading, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S rRNA cRNA (pT718S; Ambion). Following washing, the blots were digested with ribonuclease A and radioactivity associated with slots quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ). Data were expressed as relative units.
Statistical Analyses
All quantitative data were subjected to least-squares ANOVA using the general linear model (GLM) procedures of the Statistical Analysis System (SAS Institute, Cary, NC). Slot-blot hybridization or RT-PCR data were corrected for differences in sample loading using the 18S rRNA or ACTB data as a covariate. Analyses were performed within an exposure period, and tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P value of 0.05 or less was considered to be significant. Data are presented as least-square mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In study 1, exposure of neonatal sheep to EV from birth to PND 14 (period 1) inhibited development of tubular and slightly coiled endometrial glands (Fig. 1A). In Study 2, exposure to EB from PND 14 to PND 28 (period 2) or from PND 42 to PND 56 (period 3) retarded development of coiled and branched endometrial glands as assessed on PND 28 or PND 56, respectively (Fig. 1B). More details on the effects of exposure to these estrogens on uterine weight and histoarchitecture are available [14, 21].
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Semiquantitative RT-PCR Analyses
Semiquantitative RT-PCR analyses were conducted using total RNA isolated from uteri of sheep in Study 1 to determine effects of EV on intrinsic growth factor systems that are implicated as regulators of postnatal uterine development, particularly endometrial adenogenesis. The number of PCR cycles was optimized for each primer pair to ensure amplification within the linear range of detection (data not shown). Each of the primer pairs used for RT-PCR amplified a single product of the predicted size. The amplified products were sequenced to confirm identity (data not shown).
As compared to mRNA levels in uteri of CX sheep (Table 2), uteri of EV-50 sheep had substantially lower levels of FGF7 (P < 0.0009) and MET (P < 0.01) on PND 14. In contrast, the levels of INHBA mRNA were greater (P < 0.004) in the uteri of EV-50 compared CX sheep on PND 14. Levels of FGF10, FGFR2, HGF, ACVR1, ACVR2, and FST mRNA were not affected (P > 0.10) by exposure to EV.
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Identification of Estrogen-Regulated Uterine Genes by SSH
The PCR-based SSH was used to identify and clone mRNAs affected by exposure to EV during the initial budding and tubulogenic stage of endometrial adenogenesis (study 1). A differential screening procedure was performed on 400 clones from the CX-EV subtracted cDNA library to identify cDNAs representing differentially expressed mRNAs. Clones that were differentially expressed were expected to hybridize predominantly with CX cDNAs, whereas clones that hybridized with both CX and EV cDNA probes were considered to be background [24]. The differential screen yielded 26 cDNAs from the CX-EV subtracted library, and they were sized by restriction enzyme digestion and sequenced. The resulting nucleotide sequences were analyzed for similarity to all nonredundant database sequences using BLAST [26]. As summarized in Table 3, sequences of the SSH clones were highly similar (>90%) to ABCG2, CXCL10, ESR1, GSTM3, IDH1, IGFBP3, IGLV, OAS1, PECI, TEGT, and ZNF32. Many of the SSH clones were homologous to the same gene; that is, 7 of the 26 sequences matched GSTM3. Expression of ESR1 and IGFBP3 already has been shown to be reduced in the uteri of neonatal sheep exposed to estrogens [15, 21]. Two of the SSH clones had no significant sequence similarity to any known sequence in the databases.
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In Situ Hybridization and Slot-Blot Analysis of Differentially Expressed cDNAs
Effects of exposure of neonatal sheep to estrogens during three developmental periods on steady-state levels of SSH-identified mRNAs in the uterus were assessed by slot-blot hybridization analysis (Fig. 2). In situ hybridization analysis, using probes generated from SSH clones, identified which uterine cell types expressed the SSH mRNAs (Figs. 3–5). Results of ZNF32 and ABCG2 mRNA analyses are not shown, because they were below the detectable limits of in situ hybridization and slot-blot analyses (data not shown).
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The GSTM3 mRNA was observed predominantly in the endometrial LE and superficial ductal GE (sGE) (Fig. 3A). In Study 1, uterine GSTM3 mRNA levels were lower (P < 0.05) in the endometrial LE of PND-14 sheep exposed to EV from birth (Figs. 2A and 3A). In Study 2, exposure of sheep to EB from PND 14 to PND 28 reduced (P < 0.05) GSTM3 expression in the endometrial LE and sGE on PND 28, but exposure to EB from PND 42 to PND 56 did not affect (P > 0.10) GSTM3 mRNA levels on PND 56 (Figs. 2A and 3A).
The IDH1 and PECI mRNAs also were predominantly expressed in the endometrial LE (Fig. 3, B and C). In Study 1, exposure of sheep to EV from birth reduced levels of IDH1 and PECI mRNAs in the endometrial LE (Figs. 2A and 3, B and C). In contrast, exposure of sheep to EB (Study 2) did not affect (P > 0.10) uterine IDH1 and PECI mRNA levels (Figs. 2B and 3, B and C).
The IGFBP3 mRNA was observed in the periglandular stroma of the endometrium and in the myometrium but was not detected in the endometrial LE or GE (Fig. 4A). In Studies 1 and 2, exposure of neonatal sheep to either EV or EB reduced (P < 0.05) uterine IGFBP3 mRNA expression in the endometrial stroma and myometrium (Figs. 2, A and B, and 4A).
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The OAS1 mRNA was detected primarily in the endometrial LE and GE and, to a lower extent, in a subset of cells in the stroma, which morphologically appeared to be similar to immune cells, in uteri from CX sheep (Fig. 4B). In Studies 1 and 2, exposure of neonatal sheep to either EV or EB reduced (P < 0.05) uterine OAS1 mRNA expression, particularly in the endometrial LE and, if present, GE (Figs. 2, A and B, and 4B).
The TEGT mRNA was expressed in all uterine cell types but was most abundant in the endometrial LE and GE (Fig. 4C). In Study 1, exposure of neonatal sheep to EV from birth reduced (P < 0.05) uterine TEGT mRNA levels in the endometrial LE (Figs. 2A and 4C). Although exposure of neonatal sheep to EB from PND 14 to PND 28 reduced (P < 0.05) uterine TEGT mRNA levels on PND 28 (Figs. 2B and 4C), exposure to EB from PND 42 to PND 56 did not affect (P > 0.10) uterine TEGT mRNA levels on PND 56 (Figs. 2C and 4C). As in Study 1, effects of EB on TEGT mRNA in PND 28 uteri was manifest in the endometrial LE and GE (Fig. 4C).
Expression of CXCL10 mRNA was detected in cells within or near the endometrial LE and sGE, which morphologically appeared to be similar to immune cells, in uteri of CX sheep (Fig. 5A). In Study 1, exposure to EV reduced (P < 0.05) uterine levels of CXCL10 mRNA (Fig. 2A). Indeed, the numbers of CXCL10 mRNA-positive cells, although not quantified, appeared to be reduced in the endometrium of EV-exposed sheep on PND 14 (Fig. 5A). In Study 2, no effects of EB at PNDs 28 and 56 on uterine CXCL10 mRNA levels or apparent number of CXCL10-positive cells were detected (Figs. 2, B and C, and 5A).
Expression of IGLV mRNA also was detected specifically in a subset of cells within the endometrial stroma that appeared to be similar morphologically to immune cells (Fig. 5B). Slot-blot analyses of uterine total RNA did not detect any effect of estrogen treatment on IGLV mRNA levels (Fig. 2). The overall levels of IGLV mRNA in the uterus were very low. However, the number of IGLV mRNA-positive immune cells was clearly reduced in the endometrium of ewes exposed to EV from birth (Study 1) (Fig. 5B). In contrast, the number of IGLV-positive cells was not affected by exposure of neonatal sheep to EB in Study 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several of the genes (ESR1 and IGFBP3) identified by SSH already were known to be expressed in the neonatal ovine uterus and to be regulated negatively by estrogen [14, 15, 21]. Expression of the other genes identified by SSH has not been reported in the neonatal uterus of any mammal. The identified genes were expressed in a number of uterine cell types with most in the endometrial epithelia and were disrupted by estrogen in a period-dependent manner associated with either complete inhibition or retardation of endometrial adenogenesis and a reduction in overall uterine growth. The predominance of epithelial genes negatively regulated by estrogen likely results from the abundant expression of ESR1 in the endometrial epithelia during postnatal ovine uterine development [9, 11, 14, 21]. Most of the genes (OAS1, GSTM3, IDH1, PECI, and TEGT) were most abundantly expressed in the endometrial LE of CX ewes and were specifically inhibited or repressed in the LE of ewes treated with EV from PND 0 to PND 14. With the exception of OAS1, GSTM3, and IGFBP3, the other SSH-identified genes were not affected by EB exposure from PND 14 to PND 28 or from PND 42 to PND 56. Although this observation could be a result of the differences in estrogenicity of the compounds, the findings support the idea that the potential for developmental disruption is inversely related to the age or developmental state of the organ. Nonetheless, OAS1, GSTM3, IDH1, PECI, and TEGT are hypothesized to be more involved in the initial differentiation and budding of the GE from the LE that occurs primarily between birth and PND 14 and, to a degree, from PND 14 to PND 28. Furthermore, OAS1 and IGFBP3 may be involved in the tubulogenesis and coiling/branching morphogenesis that occurs primarily after PND 14 to PND 56, because EB exposure from PND 14 to PND 28 and from PND 42 to PND 56 specifically decreased OAS1 in the endometrial GE as well as LE and IGFBP3 in the stroma. Indeed, the intrinsic IGF system in the uterus appears to be an important regulatory system governing endometrial gland morphogenesis in neonatal sheep as well as humans [15]. The effects on adult reproductive function of transient exposure of the neonatal ewe to EV or EB are not known.
Mechanistic experiments on the precise roles of each candidate regulatory gene identified by SSH in the present study need to be conducted in the neonatal ewe. GSTM3 is a member of the glutathione S-transferase (GST) family that conjugates reduced glutathione to a number of exogenous and endogenous hydrophobic electrophiles. Furthermore, GST and may govern uptake and detoxification of both endogenous compounds and xenobiotics in its biological role of protecting cells from oxidative DNA damage [29]. Interestingly, exposure of the mouse uterus to estrogen increased cellular oxidative DNA damage, which was associated with a repression of GST activity [29]. IDH1 is a soluble member of the isocitrate dehydrogenase 1 (NADP+) family that functions in the citrate cycle (TCA cycle), glutathione metabolism, and reductive carboxylate cycle (CO2 fixation). Both mitochondrial and cytosolic IDH1 are involved in cellular defense against oxidative damage [30, 31]. Thus, EV repression of GSTM3 and IDH1 in the endometrial LE could predispose the epithelia to oxidative damage, thereby disrupting GE stem cell differentiation and development. PECI is a novel gene that encodes a monofunctional peroxisomal delta(3),delta(2)-enoyl-coenzyme A [32] involved in fatty acid metabolism. TEGT also is known as bax inhibitor-1 and is an intracellular, multimembrane-spanning protein that inhibits apoptosis [33]. Expression of PECI and TEGT has not been reported in the uterus.
OAS1 is a member of the 2-5A synthetase family responsible for mediating resistance to viral infection as well as proliferation, differentiation, and apoptosis of cells [34]. The effects of the estrous cycle and early pregnancy on OAS1 gene expression in the ovine uterus have been reported previously by our laboratory [35]. Interestingly, OAS1 expression in the endometrial LE was not detected in uteri from either cyclic or pregnant sheep. The biological role of OAS1 in postnatal uterine development may be mediation of cell proliferation and differentiation [36]. Another potential role for OAS1 in the nascent and developing endometrial GE is in the PRL signaling pathway. McAveney et al. [37] identified OAS1 as a protein that interacts with the human LPRLR (long form of the PRL receptor) and demonstrated that OAS1 has a role in modulating STAT1-mediated signaling by the LPRLR, which in the neonatal and adult sheep is uniquely expressed in the endometrial GE [9, 12]. Pituitary PRL regulates endometrial adenogenesis in the neonatal ovine uterus and activates STAT1, which is expressed in the developing GE [12]. Thus, OAS1 in the developing endometrial GE may regulate signal transduction pathways activated in response to PRL, which appears to be a primary regulator of endometrial gland morphogenesis in the neonatal ewe [12].
An unexpected and novel finding of the present study was the identification of CXCL10 and IGLV, because they were expressed in cells that appeared to be similar morphologically to immune cells and were reduced in the uteri of ewes exposed to EV from PND 0 to PND 14. In adult sheep, Nagaoka et al. [38] found that CXCL10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. In that study, CXCL10 (also known as IP-10) mRNA was found in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. IGLV would be a marker of immunoglobulin-producing B cells. How EV exposure lowers the number of CXCL10- and IGLV-positive immune cells in the uterus is not known. One hypothesis is that EV inhibits peripheral immune cell differentiation or, perhaps, extravasation of immune cells into the uterus by inhibiting a chemoattractant. Another hypothesis is that EV exposure directly inhibits CXCL10 and IGLV gene expression in the resident uterine immune cells or causes immune cell apoptosis. Although immune cells are present in the neonatal ovine uterus [7, 9], the present results are, to our knowledge, the first to indicate that these cells may play a biological role in uterine development and, particularly, endometrial adenogenesis, as proposed in the development of the endometrium and decidua of other mammals [39, 40].
In summary, the present results indicate that transient exposure of uteri of neonatal sheep to estrogens during critical developmental periods disrupts normal patterns of uterine gene expression in a cell type-specific manner that is associated with inhibition or retardation of endometrial adenogenesis. Future studies will aim to define the nature and function of the genes identified in these studies as potential regulators of endometrial gland morphogenesis in the neonatal ovine uterus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; tspencer{at}tamu.edu
Received: 14 April 2005.
First decision: 5 May 2005.
Accepted: 16 June 2005.
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on 2',5'-oligoadenylate synthetase expression in the ovine uterus. Biol Reprod 2001 64:1392-1399-inducible protein 10 kDa, is stimulated by IFN- and recruits immune cells in the ovine endometrium. Biol Reprod 2003 68:1413-1421This article has been cited by other articles:
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  K. Hayashi, D. W. Erikson, S. A. Tilford, B. M. Bany, J. A. Maclean II, E. B. Rucker III, G. A. Johnson, and T. E. Spencer Wnt Genes in the Mouse Uterus: Potential Regulation of Implantation Biol Reprod, May 1, 2009; 80(5): 989 - 1000. [Abstract] [Full Text] [PDF]
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 K. Hayashi, A. R O'Connell, J. L Juengel, K. P McNatty, G. H Davis, F. W Bazer, and T. E Spencer Postnatal uterine development in Inverdale ewe lambs Reproduction, March 1, 2008; 135(3): 357 - 365. [Abstract] [Full Text] [PDF]
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 J. L. Kipp, S. M. Kilen, S. Bristol-Gould, T. K. Woodruff, and K. E. Mayo Neonatal Exposure to Estrogens Suppresses Activin Expression and Signaling in the Mouse Ovary Endocrinology, May 1, 2007; 148(5): 1968 - 1976. [Abstract] [Full Text] [PDF]
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 C. J. Martyniuk, H. Xiong, K. Crump, S. Chiu, R. Sardana, A. Nadler, E. R. Gerrie, X. Xia, and V. L. Trudeau Gene expression profiling in the neuroendocrine brain of male goldfish (Carassius auratus) exposed to 17{alpha}-ethinylestradiol Physiol Genomics, November 21, 2006; 27(3): 328 - 336. [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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