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BOR - Papers in Press, published online ahead of print October 26, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046656
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BIOLOGY OF REPRODUCTION 74, 383–394 (2006)
DOI: 10.1095/biolreprod.105.046656
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Identification of Endometrial Genes Regulated by Early Pregnancy, Progesterone, and Interferon Tau in the Ovine Uterus1

C. Allison Gray 3, Colette A. Abbey 3, Phillip D. Beremand 4, Youngsok Choi 3, Jennifer L. Farmer 3, David L. Adelson 3, Terry L. Thomas 4, Fuller W. Bazer 3, and Thomas E. Spencer 2 3

Center for Animal Biotechnology and Genomics,3 Department of Animal Science and Laboratory for Functional Genomics,4 Department of Biology, Texas A&M University, College Station, Texas 77843

ABSTRACT

During early pregnancy in ruminants, progesterone (P4) from the corpus luteum and interferon tau (IFNT) from the conceptus act on the endometrium to regulate genes important for uterine receptivity and conceptus growth. The use of the uterine gland knockout (UGKO) ewe has demonstrated the critical role of epithelial secretions in regulation of conceptus survival and growth. A custom ovine cDNA array was used to identify alterations in gene expression of endometria from Day 14 cyclic, pregnant, and UGKO ewes (study 1) and from cyclic ewes treated with P4 or P4 with ZK 136,317 antiprogestin and control proteins or IFNT (study 2). In study 1, expression of 47 genes was more than 2-fold different between Day 14 pregnant and cyclic endometria, whereas 23 genes was different between Day 14 cyclic and UGKO endometria. In study 2, 70 genes were different due to P4 alone, 74 genes were affected by IFNT in a P4-dependent manner, and 180 genes were regulated by IFNT in a P4-independent manner. In each study, an approximately equal number of genes were found to be activated or repressed in each group. Endometrial genes increased by pregnancy and P4 and/or IFNT include B2M, CTSL, CXCL10, G1P3, GRP, IFI27, IFIT1, IFITM3, LGALS15, MX1, POSTN, RSAD2, and STAT5A. Transcripts decreased by pregnancy and P4 and/or IFNT include COL3A1, LUM, PTMA, PUM1, RPL9, SPARC, and VIM. Identification and analysis of these hormonally responsive genes will help define endometrial pathways critical for uterine support of peri-implantation conceptus survival, growth, and implantation.

endometrium, female reproductive tract, gene regulation, interferon, microarray, ovine, pregnancy, progesterone, uterus

INTRODUCTION

Conceptus (embryo/fetus and placental membranes) survival and growth is dependent on the uterus. During early pregnancy, the endometrium synthesizes and secretes as well as selectively transports a variety of substances, collectively termed histotroph, into the uterine lumen [1, 2]. Histotroph is a complex mixture of transport proteins, adhesion proteins, protease inhibitors, cytokines, growth factors, hormones, amino acids, and ions [3]. In laboratory rodents, several components of uterine histotroph, including leukemia inhibitory factor and calcitonin, are necessary for conceptus survival and growth and establishment of uterine receptivity [4]. In humans, histotroph also appears to be a primary source of nutrition for conceptus development during the first trimester before hematotrophic nutrition is established [5, 6]. Uterine secretions are hypothesized to be of particular importance for conceptus survival and growth in domestic animals because of the prolonged length of the peri-implantation period and superficial nature of implantation and placentation [2, 7, 8].

The uterine gland knockout (UGKO) ewe model was developed to study the role of endometrial epithelia in conceptus survival and development [9]. In this model, postnatal endometrial gland morphogenesis is inhibited by exposing neonatal ewes to a 19-norprogestin from birth to at least 8 wk of age [10, 11]. Progestin exposure specifically ablates development of the endometrial glands in the uterus without altering development of the uterine myometrium or other Müllerian duct-derived female reproductive tract structures [12, 13]. In addition to the complete absence of endometrial glands, the endometrium of UGKO ewes contains substantially less luminal epithelium because of the lack of endometrial folds [12]. Adult UGKO ewes were able to conceive but unable to establish pregnancy and exhibited recurrent early pregnancy loss [10, 12, 14, 15]. Morphologically normal blastocysts were found in the uterine flushings of bred UGKO ewes on Days 6 and 9 postmating but not on Day 14 [12, 15]. On Day 14, uterine flushings from bred UGKO ewes contained either no conceptus or a severely growth-retarded conceptus that had failed to elongate from a tubular to filamentous form [14, 15]. Available results indicate that the uterus of UGKO ewes fails to support conceptus survival and elongation because of the absence of, or a reduction in, histotroph of endometrial epithelial origin, resulting in endometrial insufficiency [14]. In sheep, conceptus elongation and trophoblast outgrowth begins on Day 12 and is completed by Day 16, which marks the beginning of implantation [16, 17]. Coincident with conceptus elongation, the trophoblast produces large amounts of IFNT (interferon tau), a Type I IFN that is antiluteolytic and the signal for maternal recognition of pregnancy in ruminants [18].

Endometrial gene expression during early pregnancy is regulated by the sequential actions of P4 from the ovarian corpus luteum and then hormones from the conceptus, such as IFNT, placental lactogen, and placental growth hormone [17, 19, 20]. In addition to the antiluteolytic effects of IFNT, a number of genes are induced or stimulated by IFNT in the endometrium, such as CXCL10, LGALS15 (galectin-15, also known as ovgal11), and WNT7A, which are implicated in conceptus development and implantation [19, 21–24]. Although commercially available and custom microarrays are available for humans, laboratory rodents, and some domestic animals (bovine and porcine), ovine microarrays are not currently available. Therefore, a custom endometrial cDNA array from Day 14 pregnant ewes was constructed and used to gain insight into hormonally regulated genes crucial for endometrial support of early conceptus survival and growth in sheep.

MATERIALS AND METHODS

Animals

Experimental and surgical procedures complied with the Guide for Care and Use of Agriculture Animals and were approved by the University Laboratory Animal Care and Use Committee and Institutional Agricultural Animal Care and Use Committee of Texas A&M University.

UGKO ewes were produced as described previously [10, 11] by implanting crossbred ewe lambs with a single Synchromate B (Sanofi, Overland Park, KS) implant within 12 h of birth and every 2 wk thereafter for a total of 8 wk. Implants were inserted subcutaneously in the periscapular area and contained approximately 6 mg of norgestomet (17{alpha}-acetoxy-11ß-methyl-19-norpreg-4-ene-3,20-dione), a potent synthetic 19-norprogestin, which was released over a 14-day period [25]. Control ewes did not receive implants.

Study 1

To synchronize estrus, adult UGKO (n = 4) and cycling crossbred ewes (n = 8) were given two i.m. injections (0700 and 1700 h) of 10 mg prostaglandin F2{alpha} (Lutalyse; Upjohn, Kalamazoo, MI) 9 days apart. Ewes were monitored daily for estrous behavior using vasectomized rams. All UGKO and one-half of the cycling ewes (n = 4) were bred when detected in estrus (Day 0) and 12 and 24 h later by intact rams of proven fertility. The remaining cyclic ewes (n = 4) were not bred to intact rams and assigned to cyclic status. On Day 14 postestrus/mating, all ewes were subjected to midventral laparotomy, and the uterine lumen was flushed with 20 ml sterile saline followed by ovariohysterectomy to obtain the uterus for collection of endometrium. Uterine flushes were analyzed using a dissecting microscope to recover conceptuses, if any, and determine their morphology.

Several sections (~1 cm) from the middle of each uterine horn were fixed in 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). The endometrium was physically dissected from myometrium for the remainder of each uterine horn and then snap-frozen in liquid nitrogen and stored at –80°C for RNA extraction. In this study, only endometrium ipsilateral to the ovary bearing the corpus luteum was used for further analysis.

Study 2

In study 2, cyclic crossbred ewes (n = 20) were checked daily for estrus and then ovariectomized and fitted with indwelling uterine catheters on Day 5 as described previously [26]. Ewes were then randomly assigned (n = 5 per treatment) to receive daily i.m. injections of P4 and/or a progesterone receptor (PGR) antagonist (ZK 136,317; Schering AG, Germany) and intrauterine (i.u.) infusions of control serum proteins and/or recombinant ovine IFNT protein in a 2 x 2 factorial as follows: 1) 50 mg P4 (Days 5–16) and 200 µg control (CX) serum proteins (Days 11–16) [P4 + CX]; 2) P4 and 75 mg ZK 136,317 (Days 11–16) and CX proteins [P4 + ZK + CX]; 3) P4 and IFNT (2 x 107 antiviral units from Days 11 to 16) [P4 + IFN]; or 4) P4 and ZK and IFNT [P4 + ZK + IFN]. Steroids were administered daily in corn oil vehicle. Both uterine horns of each ewe received twice daily injections of either CX proteins (50 µg/horn/injection) or recombinant ovine IFNT (5 x 106 antiviral units/horn/injection with CX proteins). Recombinant ovine IFNT was produced in Pichia pastoris and purified as described previously [27]. Proteins were prepared for intrauterine injection as described previously [26]. This regimen of P4 and IFNT mimics the effects of P4 and the conceptus on endometrial expression of hormone receptors and IFNT-stimulated genes during early pregnancy in ewes [21, 28, 29]. All ewes were hysterectomized on Day 16. The uterus was processed for histology and the endometrium obtained for RNA extraction as described in study 1.

Preparation of Day 14 Pregnant Ovine Endometrial cDNA Microarray

A cDNA library was prepared by Clontech (Palo Alto, CA) from total endometrial RNA pooled from the four Day 14 pregnant ewes in study 1. The directional library was synthesized using the {lambda}TriplEx2 phage vector and cDNA generated by random and oligo-dT priming of purified polyadenylated mRNA. The library possessed >2 x 106 clones with an average insert size of 1.2 kb and less than 5% of clones from genomic, mitochondrial, or ribosomal origin. The pTriplEx2 plasmid vector containing the endometrial cDNAs was excised using the Cre-lox system. Library clones were plated onto Q-trays (Genetix, Queensway, United Kingdom) containing LB agar and carbenecillin (50 µg/ml) and grown overnight. Individual clones were picked and inoculated into the wells of 384-well plates using a Q-bot (Genetix). The cDNA clones were stored at –80°C and subsequently grown in four 96-well plates for plasmid production. Approximately 5000 clones from the library were selected, sequenced, and subjected to PCR amplification for microarray printing. These plasmids were isolated from liquid bacterial cultures using a Qiagen BioRobot 3000 system and Qiagen R.E.A.L. Prep 96 BioRobot kits (Valencia, CA).

Plasmids obtained from the library were used as templates for DNA sequencing reactions. Sequencing reactions were carried out using 5N-TCCGAGATCTGGACGAGC-3N; primer and Big Dye Terminator Cycle Sequencing Ready Reactions (Applied Biosystems, Foster City, CA) in MJ PTC-200 96-well thermocyclers (Waltham, MA). Salts and unincorporated dye terminators were removed using Montage 96 Sequencing Reaction Cleanup kits (Millipore, Billerica, MA), and reaction products were analyzed on a MegaBACE automated DNA sequencer (Amersham Biosciences, Piscataway, NJ). Phred was used for base calling of sequences that were vector trimmed upstream of the EcoRI site [30, 31]. Phrap was used for clustering/contiging. BLAST searches were performed on all sequences [32, 33], and the output was parsed into tabular form. Sequences of ≥50 bp with Phred 20 quality after vector clipping were deposited in GenBank as 5N ESTs with accession numbers CD285971–CD289358.

The cDNAs were amplified by PCR using 5N-TCCGAGATCTGGACGAGC-3N as a forward primer and 5N-TAATACGACTCACTATAGGG-3N as a reverse primer. PCR conditions were as follows: 95°C for 5 min, 35 cycles of 95°C for 30 sec, 54°C for 30 sec, and 72°C for 30 sec with a final extension of 72°C for 7 min. PCR products were purified by ethanol precipitation, verified using agarose gel electrophoresis, and used to create Day 14 pregnant ovine endometrial cDNA microarrays. The concentration of these cDNAs was adjusted to approximately 100 ng/µl in 3x saline sodium citrate (SSC). An OmniGrid microarrayer (GeneMachines, San Carlos, CA) equipped with 8 Telechem (Sunnyvale, CA) SMP3 pins was used to spot the PCR products onto poly-L-lysine-coated glass slides (CEL Associates, Houston, TX) in duplicate. Slides were dried and stored desiccated until used. Glass slide microarrays were rehydrated over steam and snap dried at 95°C before UV cross-linking the DNA to the slides using a Stratalinker (Stratagene, La Jolla, CA).

Microarray Probe Labeling and Hybridization

Total RNA was isolated from endometrial samples using Trizol reagent according to manufacturer's instructions (Gibco BRL, Grand Island, NY). Polyadenylated mRNA was purified using an Oligotex mRNA Kit (Qiagen), amplified with a MessageAmp aRNA Kit (Ambion, Austin, TX), and quality and quantity determined with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). A universal control sample from the pooled Day 14 pregnant mRNA was prepared and used to compare against each of the different groups in both studies. Approximately 200 ng of amplified RNA were labeled with 3DNA Array350RP Cy3 and Cy5 kits (Genisphere, Hatfield, PA) for dual labeling (pregnant vs. UGKO, pregnant vs. cyclic, pregnant vs. P4 + CX, pregnant vs. P4 + IFN, pregnant vs. P4 + ZK + CX, and pregnant vs. P4 + ZK + IFN), according to manufacturer's instructions. Each combination of fluorescent dual labeling was performed in triplicate, including one dye swap. Probes were added to slides and coverslips affixed. Slides were then incubated overnight in a humidified hybridization chamber that was submerged in a 65°C water bath. Slides were washed and visualized following a second hybridization with the capture reagent. Fluorescent intensities of Cy3 and Cy5 were measured with an Affymetrix 428 (Santa Clara, CA) array scanner at 532 and 635 nm, respectively. After scanning, spots were aligned and analyzed with GenePix Pro 3.0 microarray analysis software (Axon Instruments, Union City, CA).

Microarray Analysis

GeneSpring 7.1 (Silicon Genetics, Redwood City, CA) was used for analysis of the microarray data. To account for dye swap, the signal channel and control channel measurements for dye swapped samples were reversed. A Lowess curve was fit to the log-intensity versus log-ratio plot. Twenty percent of the data were used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10, then 10 was used instead. To eliminate any effect of the dye swap, all genes were fit to a flat line (0.90). This eliminated approximately one-half of the genes in the microarray analyses. Lists were created from genes that passed the Student t-test with a P-value of 0.05 or less, and variances were assumed equal.

RNA Analyses

The cDNAs for CXCL10, CTGF, FTH1, IFITM3, LGALS15, PTMA, and TXN were amplified by PCR using the primers and conditions described previously and T/A cloned into pCRII (Invitrogen, Carlsbad, CA). The B2M, MIC, and STAT1 cDNAs were from previous studies [34–36]. Steady-state levels of B2M, CTGF, CXCL10, IFITM3, LGALS15, MIC, and STAT1 mRNAs in the endometrium of ewes in study 2 were quantified by slot blot hybridization as described previously [11]. Briefly, radiolabeled antisense cRNA probes were generated from linearized cDNA by in vitro transcription with [{alpha}-32P]UTP. Denatured total endometrial RNA (20 µg) from ewes in study 2 were hybridized with radiolabeled cRNA probes. To correct for variation in total RNA loading, duplicate RNA slot membranes were hybridized with a radiolabeled antisense 18 rRNA cRNA (pT718S; Ambion). After washing, the blots were digested with ribonuclease A. The radioactivity associated with each slot was quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ) and expressed as relative units.

Localization of mRNA in uterine tissue sections (5 µm) from Day 14 pregnant ewes was conducted by in situ hybridization analysis as described previously [11]. Briefly, deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized ovine cDNAs using in vitro transcription with {alpha}[35S]-uridine triphosphate. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Eastman Kodak, Rochester, NY), stored at 4°C for 1–2 wk, and developed in Kodak D-19 developer. Slides were then counterstained with Gill hematoxylin stain (StatLab, Lewisville, TX), dehydrated through a graded series of alcohol to xylene, and protected with a coverslip.

Photomicroscopy

Photomicrographs were taken using a Nikon Eclipse E1000 photomicroscope (Nikon Instruments, Inc., Melville, NY). Digital images were captured with ACT-1 2.11 (Nikon) and assembled using Adobe Photoshop 7.0 (Adobe Systems, Seattle, WA).

Statistical Analyses

Data from slot blot hybridization were subjected to least-squares ANOVA (LS-ANOVA) using the General Linear Models procedures of the Statistical Analysis System (Cary, NC). Slot blot hybridization data were corrected for differences in sample loading using the 18S rRNA data as a covariate in LS-ANOVA. Data from study 1 were analyzed for effect of status (cyclic vs. pregnant and cyclic vs. UGKO). Data from study 2 were analyzed for effects of treatment (P4 + CX vs. P4 + IFN, P4 + IFN vs. P4 + ZK + IFN, P4 + CX vs. P4 + ZK + CX, P4 + ZK + CX vs. P4 + ZK + IFN) as determined using the LSMEANS/PDIFF option of the Statistical Analysis System. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P-value of 0.10 or less was considered to be significant for analyses of slot blot data. Data are presented as the least squares mean with standard error.

RESULTS

Sequence Analysis

A total of 5145 cDNAs were sequenced in the 5N direction from a Day 14 pregnant ovine endometrial cDNA library. Sequences of ≥50 bp with Phred 20 quality after vector clipping were deposited in GenBank with accession numbers CD285971–CD289358. Bioinformatic analyses indicated that the 5N ESTs consisted of 291 clusters and 4131 singletons, giving a total of 4422 nonredundant sequences (80%). A number of sequences were abundantly present in the sequencing analyses, particularly G1P3 and LGALS15. All cDNAs were arrayed onto glass slides to create a custom 5K ovine endometrial cDNA array.

Study 1

Uterine flushes from Day 14 bred ewes contained an elongated filamentous conceptus, whereas those from Day 14 bred UGKO and nonbred cyclic ewes did not contain a conceptus (data not shown). Microarray analyses of endometrium from Day 14 pregnant, cyclic, and UGKO ewes were performed using the custom ovine endometrial 5K array (Tables 1 and 2). Microarray analyses of endometrial samples from study 1 found 23 genes that were at least 2-fold different between cyclic and UGKO ewes (15 higher in C and eight higher in UGKO) and 47 genes between Day 14 pregnant and cyclic ewes (23 higher in pregnant and 24 higher in cyclic). The number of genes different between groups takes into account genes with no homology and replicates of genes.


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  		TABLE 1. Known genes identified by microarray analysis to be 2-fold different in the endometrium of cyclic and UGKO ewes in Study One.a


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  		TABLE 2. Known genes identified by microarray analysis to be 2-fold different in the endometrium of pregnant and cyclic ewes in Study One.a

Study 2

As summarized in Table 3, microarray analyses of endometrial samples from study 2 found 1) 75 genes that were at least 2-fold different (P < 0.05) between P4 + CX and P4 + IFN (36 higher in P4 + CX and 39 higher in P4 + IFN), 2) 100 genes between P4 + ZK + CX and P4 + ZK + IFN (36 higher in P4 + ZK + CX and 64 higher in P4 + ZK + IFN), 3) 180 genes between P4 + IFN and P4 + ZK + IFN (83 higher in P4 + IFN and 97 higher in P4 + ZK + IFN), and 4) 125 genes between P4 + CX and P4 + ZK + CX (58 higher in P4 + CX and 67 higher in P + ZK + CX). The number of genes different between groups takes into account genes with no homology and replicates of genes.


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  		TABLE 3. Known genes identified by microarray analysis to be 2-fold different in the endometrium of ewes in Study Two.a


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  		TABLE 3. Continued

Common known genes identified in both studies 1 and 2 are presented in Figure 1 as Venn diagrams. Twelve genes were regulated by P4 and pregnancy in the endometrium (Fig. 1A), and 14 genes were regulated by IFNT and pregnancy (Fig. 1B). In study 2, 12 genes were regulated by P4 and IFNT (Fig. 1C).


Figure 1
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  		FIG. 1. Venn diagram illustrations of common genes from the microarray experiments. A) Number of common genes that differed at least 2-fold in the endometrium of Day 14 pregnant versus cyclic ewes in study 1 and P4 + CX versus P4 + ZK + CX ewes in study 2. This compilation reveals genes regulated by progesterone. B) Number of common genes that differed at least 2-fold in the endometrium of Day 14 pregnant versus cyclic ewes in study 1 and P4 + IFN versus P4 + CX ewes in study 2. This compilation reveals genes regulated by IFNT. C) Number of common genes that differed at least 2-fold in the endometrium of P4 + IFN versus P4 + CX ewes and P4 + CX versus P4 + ZK + CX ewes in study 2. This compilation reveals genes regulated by both progesterone and IFNT

Validation of Microarray Results

Many of the genes expressed higher in endometrium from Day 14 pregnant ewes (study 1) or IFNT-infused ewes (study 2) were genes previously known to be induced or increased by IFNT from the conceptus in the endometrium of sheep during early pregnancy or by Type I IFNs in other cell types (Tables 1 and 2). Known IFNT-stimulated genes identified by microarray include B2M [34], CXCL10 [24], G1P3 [37], IFIT1 [38], IFITM1 [39], ISG12 [21], LGALS15[22], MIC [34], MX1 [40], RSAD2 (viperin or cig5) [41], and STAT1 [35]. Identification of these genes in endometria from pregnant and cyclic ewes and P4 + CX and P4 + IFN ewes validates, in part, the microarray results.

Steady-state levels of CXCL10 and LGALS15 mRNA in endometria from ewes in study 1 were determined by slot blot hybridization analysis (Fig. 2). Expression of LGALS15 mRNA was higher (P < 0.01) in pregnant than cyclic or pregnant than UGKO endometria but not different (P > 0.10) between endometria from cyclic and UGKO ewes (Fig. 2A). Similarly, CXCL10 mRNA was higher (P < 0.10) in pregnant than cyclic or pregnant than UGKO endometria (Fig. 2B) but not different (P > 0.10) in endometria from cyclic compared to UGKO ewes.


Figure 2
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  		FIG. 2. Steady-state levels of LGALS15 (A) and CXCL10 (B) mRNAs in endometrium from Day 14 pregnant, cyclic, and UGKO ewes from study 1. Endometrial mRNA levels were determined by slot blot hybridization analysis (see Materials and Methods). Data are presented as least squares mean relative units ± SEM

Steady-state levels of B2M, CTGF, CXCL10, IFITM3, MIC, and STAT1 mRNAs in endometrium from ewes in study 2 are presented in Figure 3. All of these mRNAs, except for CTGF, were 2-fold or greater in microarray analyses of endometrium from P4 + IFN over the P4 + CX ewes as well as in endometria from P4 + ZK + IFN over the P4 + ZK + CX ewes. Steady-state levels of B2M, CXCL10, IFITM3, MIC, and STAT1 mRNAs were higher (P < 0.10) in endometria of ewes receiving intrauterine infusions of IFNT in both P4 and P4 + ZK treatment groups. In contrast, CTGF mRNA was most abundant in the P4 + ZK + IFN group (P4 + ZK + IFN vs. P4 + IFN, P < 0.06). These results parallel findings from the microarray analyses.


Figure 3
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  		FIG. 3. Steady-state levels of CXCL10 (A), IFITM3 (B), B2M (C), MIC (D), STAT1 (E), and CTGF (F) mRNAs in endometrium of ewes in study 2. Endometrial mRNA levels were determined by slot blot hybridization analysis (see Materials and Methods). Data are presented as least squares mean relative units ± SEM

Localization of mRNAs in the Endometrium of Day 14 Pregnant Ewes

As illustrated in Figure 4, LGALS15 mRNA was detected solely in the endometrial luminal epithelium and superficial ductal glandular epithelium of uteri from Day 14 pregnant ewes. CTGF mRNA was detected in glandular epithelium as well as immune-like cells within the stroma. CXCL10 mRNA was detected only in immune-like cells throughout the endometrial stroma. FTH1 mRNA was expressed by a number of cell types, including luminal epithelium, glandular epithelium, immune-like cells, and stroma, but was most abundant in the luminal epithelium. Both IFITM3 and PTMA mRNA were expressed predominantly in the stratum compactum area of the endometrial stroma adjacent to the luminal epithelium. TXN mRNA was expressed at low levels by luminal epithelium and superficial ductal glandular epithelium.


Figure 4
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  		FIG. 4. In situ hybridization analysis of LGALS15, CTGF, CXCL10, IFITM3, FTH1, PTMA, and TXN mRNAs in the uterus of Day 14 pregnant ewes. Cross sections of the uterine wall were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized plasmid cDNA clones. Hybridized sections were digested with RNase A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under bright-field or dark-field illumination. LE, Luminal epithelium; GE, glandular epithelium; sGE, superficial ductal GE; S, stroma. Original magnification x40

DISCUSSION

The transcriptional profiling experiments using custom ovine endometrial cDNA arrays were effective in identifying genes in the endometrium of early pregnant ewes that are regulated by pregnancy status as well as P4 and/or conceptus IFNT. The identified genes are candidate regulators of uterine receptivity as well as conceptus growth and development. As observed in transcriptional profiling studies of human endometrium (for review, see Horcajadas et al. [42]), expression of many genes was increased and decreased in Day 14 endometrium during pregnancy establishment in the ovine uterus (Table 2). As expected, many of the 23 transcripts increased in the endometria of Day 14 pregnant as compared to cyclic ewes were encoded by known IFNT-stimulated genes, including B2M, CTSL, G1P3, IFI27, IFIT1, IFITM3, LGALS15, MX1, and RSAD2. Several of those genes, including CTSL, CXCL10, and LGALS15, are implicated in endometrial regulation of conceptus implantation [22–24, 43]. However, several other genes whose expression increased, such as GRP and HSGP2, are not classical Type I IFN-stimulated genes but are implicated in implantation in sheep and mice [44–46]. The genes whose expression decreased in the endometria of Day 14 pregnant as compared to cyclic ewes have not been previously reported in the ovine uterus, but results are consistent with the idea that endometrial responses to pregnancy include both activation and deactivation of gene expression during conceptus implantation [42, 47].

During the peri-implantation period of pregnancy, gene expression in the endometrium of ruminants is programmed primarily by P4 from the ovary and IFNT from the conceptus [20, 48, 49]. Progesterone is unequivocally required for maternal support of conceptus survival and development [48]. A comparison of endometrium from P4 + CX and P4 + ZK + CX ewes revealed P4-increased genes, including CIRH1A, COL3A1, COX2, CYB, HIF1A, CTSL, LC3, LGALS15, PEB4, PFN, POSTN, PPIA, RHOBTB3, RPL12, RPL23A, STAT5A, and TIMP2, and P4-decreased genes, including CTGF, DNPEP, DOCK9, FAM14B, G1P3, HSPCB, IFI27, IFIT1, IFITM3, MALAT1, MX1, QKI, RPS14, RPS24, RSAD2, TAGLN, and TLK1. Many of the P4-regulated genes identified in the present study have not been reported in the ovine uterus. Similar to findings in the murine and human uterus [42, 50], P4 also decreased expression of many genes in the endometrium of the ovine uterus. The complex responses of individual endometrial cell types in the uterus to P4 are regulated by PGR expression [19, 20] and highlighted by the differences in expression of the limited number of genes analyzed in the present study. In both cyclic and pregnant ewes, continuous exposure of the endometrium to P4 for 8–10 days represses PGR gene expression in the endometrial epithelia. During the estrous cycle and pregnancy, the PGR is expressed in all uterine cell types on Day 6 but restricted to stromal cells throughout pregnancy after PGR is lost from luminal epithelium and glandular epithelium on Days 11 and 13, respectively [49, 51]. The loss of PGR in the endometrial luminal epithelia and then glandular epithelia is associated with reprogramming of gene expression patterns in the endometrium [20, 49]. For example, the expression of CTSL and LGALS15 mRNA increases in the endometrial luminal epithelium and superficial glandular epithelium between Days 10 and 12 and can be induced by P4 in the endometrium [22, 43], which is correlated with the loss of PGR in those epithelia [51]. Similarly, COX2, SPP1, and SERPIN (also known as uterine milk protein) are other examples of P4-induced genes whose induction in the endometrial luminal epithelium or glandular epithelium is associated with PGR loss [26, 52, 53]. SPP1 and SERPIN were not identified in the present study because they are not expressed in the endometrium until after Day 14 of pregnancy [54, 55]. Interestingly, a number of the P4-decreased genes identified in study 2 are also classical IFN-stimulated genes, including FAM14B, G1P3, IFI27, IFIT1, IFITM3, MX1, and RSAD2. Clearly, P4 and IFNT have complex and complementary actions on a number of genes in the ovine endometrium during early pregnancy.

Although usually associated with antiviral responses, IFN-stimulated genes are common to pregnancy in many mammals, including humans, primates, rats, mice, ruminants, and pigs [4, 20, 49]. IFNT, the signal for maternal recognition of pregnancy in ruminants, is secreted by the ovine conceptus trophectoderm between Days 10 and 20 of pregnancy, with maximum production by the trophoblast on Days 15 and 16 [56–58]. Previous studies found that IFNT stimulates expression of most genes in the endometrial stroma and middle to deep glandular epithelium, such as B2M and MIC [34], OAS1[29], and STAT1 [35]. In the present studies, transcriptional profiling of endometrium identified a number of known IFNT-stimulated genes, including B2M, CTSL, G1P3, IFITM3, IFIT1, and LGALS15. Studies identified other IFNT-stimulated genes, including CXCL10 expressed by immune cells and IFITM3 expressed by stroma of the endometrium as shown previously [24, 39]. Study 2 also identified a number of novel IFNT-stimulated genes, including aut2b2, COL4A3BP, DDX5, DNPEP, GRP, HIF1A, SEPP1, and TAGLN. On the other hand, a number of IFNT-decreased genes were also identified, including COL3A1, POSTN, S100A2, SPARC, and TLN1. A number of these genes appear to be coregulated by P4 and IFNT in the ovine endometrium. For instance, P4 induces and then IFNT stimulates CTSL and LGALS15 expression in the endometrial luminal epithelium and superficial glandular epithelium during early pregnancy in the ovine uterus [22, 43].

A primary objective of study 1 was to identify genes different in the endometrium of fertile cyclic and infertile UGKO ewes, which would presumably identify genes abundantly expressed in the glandular epithelium. Similar to a previous study [11], several genes were found to be different in endometria of cyclic and UGKO ewes (Table 1), but very few of the known genes encode secreted proteins or were expressed in the endometrial glands. Indeed, the majority of the genes different in the endometrium of cyclic and UGKO ewes were immunoglobulin genes, likely because of the large numbers of immune cells present in the endometrium [59]. Thus, the UGKO endometrium may contain different populations or altered numbers of immune cells relative to normal ewes, which could also be involved in the etiology of recurrent early pregnancy loss observed in the UGKO ewes as hypothesized in humans [60]. Unfortunately, the nature of the immune cells in the uterus and their role during early pregnancy in the ewe are not well studied. The inability of the present study to identify genes that are deficient in the UGKO endometrium could be due to the sensitivity of the microarray analysis as well as the use of whole endometrium for the studies. Profiling of individually isolated cell populations, such as luminal epithelium, glandular epithelium, stroma and immune cells, from the endometrium may be useful in future studies. Further, the recurrent early pregnancy loss observed in the UGKO ewe could be due to the lack of certain histotroph components that are selectively transported into the uterine lumen by the endometrial glands from serum transudate as opposed to specific secretory products encoded by genes expressed in the glands [61]. Consequently, future studies should analyze the composition of the histotroph using an integrated proteomics and metabolomics approach to uncover differences between the intraluminal components of the normal fertile and infertile UGKO uterus.

In summary, these transcriptional profiling studies identified a number of genes that are regulated by pregnancy, P4, and IFNT in the endometrium of the ovine uterus. Several of the identified genes may be conserved in the uterus of other mammals and involved in establishment of uterine receptivity and conceptus implantation. Future studies of the identified genes in cyclic and pregnant ewes during the peri-implantation period are expected to unravel the complex hormonal, cellular, and molecular mechanisms regulating gene expression and endometrial function in the ovine uterus during early pregnancy. The genes identified in the present study are regulated by P4 and conceptus IFNT and transcribed in the endometrium during the period in which the majority of pregnancy loss occurs in livestock as well as humans [2, 62, 63] and thus are likely to be important for conceptus survival and growth during the peri-implantation period of pregnancy [17, 19].

ACKNOWLEDGMENTS

The authors would like to thank members of the laboratory for assistance with animal husbandry and surgery as well as the expert technical assistance of Josh Munson and Beth Riedel from the Laboratory for Functional Genomics.

FOOTNOTES

1 Supported by National Research Initiative Competitive grant 2001–35203–10700 from the USDA Cooperative State Research, Education, and Extension Service and NIH grants HD32534 and P30ES09106. Back

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 Back

Received: 14 August 2005.

First decision: 13 September 2005.

Accepted: 24 October 2005.

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