MOLECULAR REPRODUCTION AND DEVELOPMENT 65:9–18 (2003) Characterization of Gene Expression Profiles in Early Bovine Pregnancy Using a Custom cDNA Microarray HIROKO ISHIWATA,1 SUSUMU KATSUMA,2 KEIICHIRO KIZAKI,1 OSMAN V. PATEL,1 HIROKO NAKANO,1 TORU TAKAHASHI,1 KEI IMAI,1 AKIRA HIRASAWA,2 SATOSHI SHIOJIMA,2,3 HIROSHI IKAWA,2 YASUHITO SUZUKI,2 GOZOH TSUJIMOTO,2 YOSHIAKI IZAIKE,1 JUNICHI TODOROKI,4 AND KAZUYOSHI HASHIZUME1* 1 Laboratory of Reproductive Biology and Technology, Department of Developmental Biology, National Institute of Agrobiological Sciences, Ibaraki, Japan 2 Department of Molecular Cell Pharmacology, National Center for Child Health and Development, Tokyo, Japan 3 Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Mie, Japan 4 Kagoshima Prefectural Cattle Breeding and Developmental Institute, Kagoshima, Japan ABSTRACT Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placentalspecific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/ endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy. Mol. Reprod. Dev. 65: 9–18, 2003. embryo and maternal endometrium. The cascade of implantation is orchestrated by various proteins, cytokines, and growth factors synthesized and secreted by the trophoblast cells that systematically modulate maternal anatomy, endocrinology, immunology, and physiology to create an environment conducive to fetal development and survival (Jauniaux et al., 2000). In cattle, maternal and fetal tissues fuse at discrete areas to form a placentome. Each disc-shaped placentome consists of a fetal component, the cotyledon, and a maternal component, the caruncle (Wooding and Flint, 1994). Embryonic mortality accounts for major reproductive wastage in farm animals (Cross et al., 1994). Moreover, 80% of this mortality is attributed to a dysfunctional placenta. To reduce these early losses, as well as to improve the reproductive efficiency of cattle by application of technologies such as artificial insemination, embryo transfer and cloning, requires a precise knowledge of the intricate dialogue between the feto– materno–placental unit in the establishment and maintenance of pregnancy. However, little is known about the complex molecular regulation of placental development, especially in cattle. Thus, elucidating the mechanisms that control normal placentation is ß 2003 Wiley-Liss, Inc. Key Words: normalized library; hit-picking; uterus; placenta; gestation INTRODUCTION An essential feature of mammalian pregnancy is the formation of a specialized organ, the placenta, which bridges the developing fetus and the dam. Establishment of the placenta involves a series of events known as implantation, when fetally derived placental cells (trophoblast) invade a modified layer of maternal endometrium. It is a complex process and successful implantation requires appropriate communication between the ß 2003 WILEY-LISS, INC. Hiroko Ishiwata and Susumu Katsuma have contributed equally to this work. Grant sponsor: Organized Research Combination System (from the Education, Science and Technology Agency); Grant sponsor: Bio-Oriented Technology Research Advancement Institution, Japan. *Correspondence to: Kazuyoshi Hashizume, Laboratory of Reproductive Biology and Technology, Department of Developmental Biology, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukubacity, Ibaraki 305-8602, Japan. E-mail: kazuha@affrc.go.jp Received 8 October 2002; Accepted 18 November 2002 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.10292 10 H. ISHIWATA ET AL. important for identification of genes regulating implantation and placentogenesis as well as understanding the pathophysiology of the compromised placenta. Earlier techniques, such as Northern and Southern blotting, and later, the S1 nuclease protection method, had major limitations in that only a restricted number of genes could be studied concurrently, and they provided no insights into differential gene expression. However, the development of cDNA and oligonucleotide array techniques in the last decade has provided an opportunity to study expression levels in parallel, thus yielding static as well as dynamic information for hundreds to thousands of genes simultaneously. Applications of cDNA microarray technology have varied from studying gene expression in yeast under different environmental stress conditions (Schena et al., 1995) to the comparison of gene expression profiles for tumors from cancer patients (DeRisi et al., 1996; Ahrendt et al., 1999). Moreover, genome-wide expression profiling has recently been employed to study the multigenic regulation of implantation in human and mice (Tanaka et al., 2000; Yoshioka et al., 2000; Kao et al., 2002), as well as, trophoblast differentiation in humans (Aronow et al., 2001; Chen et al., 2002). In the present study, we report on the establishment of a bovine placental-specific cDNA microarray and the profiles of genes expressed in early pregnancy. MATERIALS AND METHODS Animals and Tissues Endometrial (caruncular and intercaruncular endometrium) and placental tissues (cotyledonary and intercotyledonary fetal membrane) for establishing cDNA libraries was collected separately from Japanese Black cows on days 0 and 10 of the estrous cycle, and days 30, 60, 100, and 245 of gestation (day 0 was referenced as the day of standing estrus). For hybridization, endometrial and placental tissues were also collected separately on day 13 (n ¼ 2) of the estrous cycle and days 56 (n ¼ 2), 59, and 64 of gestation. The endometrial and placental tissues were collected and separated as detailed by Nikitenko et al. (1998) and Regnault et al. (2002), and illustrated schematically by Schauser et al. (1998). Total RNA was isolated from these tissues by using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions and used for raising the array, hybridization, and RT-PCR analysis. Construction of a Bovine Placental/Endometrial cDNA Library Poly (A)þ RNA, prepared from total RNA using the Oligotex-dT30 RNA isolation kit (Takara, Kyoto, Japan), was employed to construct a phage cDNA library with the ZAP Express Vector Kit (Stratagene, San Diego, CA). cDNA fragments ranging from 500 to 2,500 bp were recovered by the GeneClean method (BIO 101, Vista, CA) following agarose gel electrophoresis. A phagemid cDNA library was excised in vivo from the phage library with a helper phage (ExAssist, Stratagene). Construction of a Normalized cDNA Library by Hit-picking About 5,000 of 1,200,000 clones were randomly selected and those that were redundant eliminated by the hit-picking method. Finally a spot array of about 4,000 genes was established as described in detail elsewhere (Katsuma et al., 2001). In brief, bacterial colonies were randomly inoculated into 96-well plates for culture by using a colony picker. PCR was performed in a 96-well format for preparing the cDNA fragments. Macroarray filters were generated by using the Biomek 2000 HDRT (High-Density Replicating Tool, 96 pin plate) system (Beckman Coulter, Fullerton, CA). After spotting, the filters were washed once in 2
SSC (2
saline-sodium citrate solution: 0.3 M NaCl, 15 mM sodium citrate, pH 7.0) followed by UV cross-linking (120 mJ) using a UV Stratalinker 1800 (Stratagene). The hybridization probe was prepared by using the DIG DNA labeling kit (Roche Diagnostics, Tokyo, Japan). After color development, macroarray membranes were scanned (GT-9000, Epson, Tokyo, Japan) and abundant clones were removed robotically (Biomek 2000 workstation, Beckman Coulter). Rearranged clones were stored at 808C as a normalized library. cDNA Microarray Analysis cDNA clones selected by the hit-picking method were amplified by PCR and spotted onto glass slides robotically. Simultaneously, the clones were sequenced by using the MegaBACE 1000 DNA Sequencing system (Amersham Pharmacia Biotech, Piscataway, NJ). The sequenced clones were compiled and annotated by BLAST, and the clone that was positioned at the top of the hierarchical tree of genes by GenBank database matching was selected. Two micrograms of poly (A)þ RNA from endometrium taken either during the estrous cycle or pregnancy was reverse transcribed with Cy3- or Cy5-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) for hybridization of probes. Following a 2-h incubation, the labeled probes were concentrated in a Microcon filter device (Millipore, Bedford, MA), diluted in 15 ml hybridization solution (3.4
SSC containing 0.3% SDS, 20 mg poly(A) DNA, and 20 mg yeast RNA), and applied to the microarray. After overnight incubation at 658C, the slides were washed twice with 2
SSC containing 0.5% SDS for 5 min at room temperature, once with 0.2
SSC containing 0.5% SDS at 408C for 3 min, and finally with 0.2
SSC for 3 min. Slides were dried by centrifugation at low speed. Hybridization images were scanned by GenePix 4000A (Axon Instrument, Union City, CA) and analyzed by GenePix Pro3.0 software. All experiments were performed in duplicate with reversed fluorescent labels; for example, first, estrous cycle endometrial RNA was labeled with Cy3 and pregnant endometrial RNA with Cy5 as a counterpart of hybridization. Then reverse labeling with fluorescence was done, and the difference between Cy3 and Cy5 was evaluated. The intensity of GENE EXPRESSION PROFILES IN EARLY BOVINE PREGNANCY 11 TABLE 1. Primers Utilized for RT-PCR Gene PAG-1 PL PRP-1 GRP Calbindin STC-1 GAPDH Forward primers (50 –30 ) CACCATTGGACCACCCCC CAACCTACTAGTCCATCTCCCCATCAGCAGCAGT ATCATAGAATTCACGGTCAACAGGAGTCCTCACC GAAATGGGCAGCCGCGAGGTCTC GAGTGCCAAAAAGTCTCCAGAAGA GTTATGGTCCAAAACTCAGCAGTGATT CCTTCATTGACCTTCACTACATGGTCTA fluorescence was expressed as a ratio; either Cy3/Cy5 or Cy5/Cy3. RT-PCR Analysis The relative levels of expression of pregnancyassociated glycoprotein-1 (PAG-1), placental lactogen (PL), prolactin-related protein-1 (PRP-1), Gastrinreleasing peptide (GRP), vitamin D-dependent calcium binding protein (Calbindin), and stanniocalcin-1 (STC-1) were determined by RT-PCR. Bovine GAPDH was used as a positive control for the PCR. Total RNA (2 mg in 11 ml) was used for reverse transcription and template cDNA was synthesized using Oligo dT primer and 200 U Superscript II reverse transcriptase. PCR reaction mixtures containing cDNA template (1 ml), 20 mM forward and reverse primers (0.4 ml each, Table 1), 2 mM deoxynucleotide triphosphate mixture (2 ml), 100 mU AmpliTaq gold DNA polymerase (0.1 ml), 25 mM MgCl2 (1.2 ml) and double-distilled water (12.9 ml), were placed in a thermal cycler (MJ Research, Inc., Watertown, MA), and subjected to the following conditions: denaturation (at 958C for 30 sec), annealing (PAG-1; 608C for 30 sec, PL; 658C for 30 sec and rest; 558C for 30 sec) and extension (728C for 1 min). PCR products were separated on agarose gels. PCR product size (bp) Reverse primers (50 –30 ) CACTGGGTAGTTGATGCCGTT CATACAAAGCGGCCGGGAGACCCATTACACCCAAACAT GTCATAGTCGACAATTTCAGGTAGCCCGCTGTGG GTCAGTACAGCTGGGGGTTCC CACTGGGATATCTTTTTCACC CTAGGCACTCTCCTGGGAGGTG GCTGTAGCCAAATTCATTGTCGTTACCA 745 867 820 402 237 744 857 RESULTS AND DISCUSSION A phage cDNA library containing approximately 1.2
106 independent clones was established from bovine endometrial and placental RNA. Of these, only 0.4% (about 5,000) were chosen from the phage cDNA library that was used for the hit-picking normalization (Katsuma et al., 2001). The hit-picking results showed about 17% redundant clones (845 clones) that were eliminated from the 4,800 gene cluster, leaving 3,955 genes that were finally spotted onto one glass slide. The efficiency was confirmed by calbindin and PAG-1, 22/62 (actual number of clones spotted on glass/expected number of clones in a cluster library) and 48/77 clones were eliminated, respectively. The efficiency of elimination was not higher than that reported previously (Katsuma et al., 2001), as we had only used about 5,000 randomly selected clustered genes in this study for hit-picking normalization. Although, it is a fairly multifaceted system compared to the conventional normalization method described by Bonaldo et al. (1996), it does facilitate the visual removal of highly expressed genes, thus selectively leaving less-abundant genes within the normalized library. In addition, it seems easier to improve the efficiency by using many more clones from the original library. TABLE 2. Classification of Genes in the Bovine Endometrial/placental cDNA Array Number of Genes Category no. 1 2 3 4 5 6 7 Sum Gene name Cell and tissue structural dynamics Intercellular communication Intracellular metabolism Cell cycle and apoptosis Regulation of gene expression EST/unknown Uncomplemented gene Number of clonesa Identifiedb Up-regulatedc Down-regulatedd 439 728 1011 31 189 974 583 3955 108 221 265 26 113 617 583 1933 7 44 8 1 5 12 30 107 37 117 113 15 66 399 324 1071 The EST/unknown category contains genes that had limited matching between sequenced clones and those in the GenBank database. The dynamic expression of individual genes was compared between uterine tissue obtained on day 13 of the estrous cycle and uterine/placental tissue of about day 60 gestation. a Number of clones spotted onto the slide. b Number of genes annotated by BLAST. The differential expression value represents the fold change of gene expression between the two groups. Differential expression values of >2 (cUp-regulated genes) and <0.5 (dDown-regulated genes) are considered to represent equivalent gene expression. 12 H. ISHIWATA ET AL. TABLE 3. Genes Up-regulated During Early Gestation in the Bovine Endometrium/Placentome Ratio (pregnancy/estrous) Accession no. Gene name Caruncle Intercaruncle Category no. AF020511 AF192333 AF192336 AF192334 AF192332 M73962 AF020509 J02840 J02944 AW465434 AF059507 L06151 AW465455 L12711 M76478 M81129 NM_012193 AF020514 NM_000975 NM_001006 AW417930 L22095 AF192338 AJ004935 Bos taurus PAG-9 Bos taurus PAG-16 Bos taurus PAG-19 Bos taurus PAG-17 Bos taurus PAG-15 Bos taurus PAG-1 Bos taurus PAG-7 Bos taurus PL Bos taurus PRP-1 Bos taurus cDNA clone BP230019A20B3 50 Bos taurus epidermal fatty acid-binding protein Bos taurus PAG-2 Bos taurus cDNA clone BP230019B10D6 50 Homo sapiens transketolase Cow insulin-like growth factor binding protein-3 Cow superoxide dismutase Homo sapiens frizzled homolog 4 Bos taurus PAG-12 Homo sapiens ribosomal protein L11 Homo sapiens ribosomal protein S3A Bos taurus cDNA 50 Bos taurus uterine milk protein Bos taurus PAG-21 Urechis caupo cytoplasmic intermediate filament protein Bos taurus thrombin inhibitor Ovine PAG-1 Oryctolagus cuniculus sodium-dependent multi-vitamin transporter Bos taurus PAG-5 Bovine endothelin ETB receptor Bos taurus fibronectin precursor Bos taurus p97 Bos taurus PRP-4 Bos taurus cDNA clone BP230013A20A12 50 Bos taurus PAG-8 Bovine galactose-binding lectin Human mariner-like element-containing mRNA, clone pcHMT1 Human 1-phosphatidylinositol-4-phosphate 5-kinase isoform C Homo sapiens tropomyosin 2 beta Homo sapiens cDNA clone IMAGE:1368527 Homo sapiens cDNA DKFZp434D146 Homo sapiens putative transcription factor Pig nonhistone protein HMG1 Homo sapiens nucleophosmin phosphoprotein Bos taurus PAG-13 Human calmodulin Homo sapiens DNA-binding zinc finger Homo sapiens alpha-tubulin isoform 1 Bos taurus cDNA clone BP230017B20B8 50 Bos taurus PAG-6 Bos taurus PAG-10 Human thymosin beta-10 Bos taurus PAG-11 Bos taurus PRP-2 Bovine estrogen sulfotransferase C familiaris TRAM-protein Bos taurus complete mitochondrial genome Danio rerio cDNA clone RZPD clone CHBOp576O20201Q3 Bovine osteonectin Bos taurus ferritin H subunit Homo sapiens stanniocalcin Ovine Gastrin-releasing peptide 10.35  2.03 9.84  1.94 7.55  2.03 6.58  3.03 6.02  2.09 5.65  2.33 5.35  2.78 4.88  1.77 4.50  1.80 4.28  1.41 3.81  1.17 3.72  1.62 3.71  1.16 3.69  0.98 3.64  1.14 3.30  1.06 3.29  0.86 3.28  1.55 3.20  0.81 3.19  0.83 3.14  1.19 3.05  1.65 3.03  1.55 2.99  1.22 0.97  0.52 1.18  0.75 1.00  0.66 1.02  0.59 0.96  0.51 0.86  0.45 1.03  0.70 1.04  0.61 0.95  0.54 0.96  0.52 1.17  0.83 0.90  0.57 1.40  0.78 0.95  0.42 1.47  0.65 0.91  0.52 1.03  0.57 1.09  0.71 14.22  4.66 0.90  0.52 1.62  0.71 17.89  9.73 0.81  0.51 1.14  0.67 2 2 2 2 2 2 2 2 2 6 2 2 6 2 2 2 2 2 3 3 6 2 2 1 2.83  0.94 2.81  0.71 2.66  0.67 0.82  0.37 0.75  0.28 1.03  0.70 2 2 2 2.60  1.05 2.57  0.74 2.47  0.74 2.43  0.56 2.33  0.79 2.32  0.58 2.27  0.92 2.24  0.60 2.18  0.75 0.93  0.52 0.94  0.44 2.53  1.25 0.86  0.48 0.99  0.52 1.05  0.49 1.15  0.92 1.01  0.49 1.39  0.60 2 2 1 4 2 6 2 1 6 2.18  1.04 1.88  1.11 2 2.17  0.64 2.15  0.32 2.15  0.62 2.13  0.55 2.13  0.75 2.13  0.59 2.13  0.79 2.13  0.54 2.11  0.71 2.10  0.62 2.09  0.55 2.09  0.30 2.08  0.30 2.08  0.28 2.06  0.84 2.05  0.44 2.03  0.56 2.00  0.42 1.54  1.64 1.35  0.48 1.03  0.54 1.19  0.60 1.07  0.47 0.86  0.48 0.81  0.33 1.25  0.49 0.89  0.61 0.80  0.45 0.80  0.38 1.09  0.47 1.07  0.52 0.83  0.48 1.19  0.78 1.03  0.44 0.94  0.40 0.70  0.23 0.86  0.44 0.84  0.35 4.04  0.58 4.39  1.81 1 6 6 5 5 5 2 2 5 1 6 2 2 1 2 2 2 5 3 6 1.27  0.44 1.17  0.32 1.12  0.30 1.12  0.40 2.11  1.25 3.23  2.08 7.46  3.82 8.28  4.06 1 2 2 2 D55670 M73961 AF080067 AF020507 D10989 AF136453 D84515 M33269 AW463583 AF020510 X14330 U48696 S78798 NM_003289 AA837482 AL117429 AJ009770 M21683 U89321 AF192330 D45887 AB017493 AF081484 AW465087 AF020508 AF020512 S54005 AF020513 M27239 M54942 X63678 V00654 AI816672 J03233 AB003093 NM_003155 S75723 (Continued) GENE EXPRESSION PROFILES IN EARLY BOVINE PREGNANCY 13 TABLE 3. (Continued ) Ratio (pregnancy/estrous) Accession no. AJ388516 L10325 AF102850 M57446 X59767 AW336014 AC005027 AA933095 NM_006429 NM_001647 U16239 AF013215 AF083243 X03205 Gene name Canis familiaris ribosomal protein L27 Cow glutathione peroxidase plasma isoform Homo sapiens dolichyl-phosphate beta-glucosyltransferase Porcine antileukoproteinase Bovine gene for brain ribonuclease Bos taurus cDNA 50 Homo sapiens BAC clone GS1-512121 from 7p14–p12 Brugia malayi cDNA clone SWBmL3SA008 50 or 30 Homo sapiens chaperonin containing TCP1, subunit 7 Homo sapiens apolipoprotein D Bos taurus betaA inhibin/activin Bos taurus ribosomal protein S2 Homo sapiens HSPC025 Human 18S ribosomal RNA Caruncle Intercaruncle Category no. 1.04  0.29 0.94  0.31 0.80  0.39 5.55  1.74 2.14  0.50 4.48  1.47 3 2 2 0.78  0.30 0.76  0.34 0.60  0.22 0.55  0.13 2.96  1.93 2.79  1.93 2.01  0.98 3.68  1.88 2 3 6 6 0.55  0.32 4.22  1.91 6 0.53  0.14 2.36  1.37 3 0.46  0.19 0.46  0.12 0.41  0.10 0.39  0.17 0.36  0.16 2.03  0.81 5.01  2.31 2.00  0.37 2.10  1.11 2.19  1.31 2 2 3 2 3 The array contained many genes that after sequence annotation were found to be analogous; therefore the average of these was used as the representative value for the annotated gene. The experiment was repeated at least twice with reverse labeling of Cy3 and Cy5 in four different animals. More than twofold differences are shown in expression values between the estrous cycle (day 13) and gestation (day 56–64). Category number is a general descriptor or gene function as indicated in Table 2. Values are mean  SD (n ¼ 4). The technical variation of hybridization caused either by the nature of the fluorescence or labeling procedure was examined by reverse labeling with Cy3 and Cy5. The endometrial samples from the estrous cycle were used for verification and were done in duplicate with reverse labeling. All values varied within the 50–200% range in comparison with the theoretical value (100%) (data not shown). Therefore, differences in levels of expression either under 50% or over 200% were taken to be significant. Comparison of placental and endometrial tissues showed a high and reproducible correlation (r ¼ 0.9003) similar to that of an earlier report (Yang et al., 2002). A total of 1,933 genes were identified from the 3,955 spotted clones onto the microarray slide. These individual genes were clustered into six overall functional categories as described by Aronow et al. (2001) and shown in Table 2; these categories were based on biological functions involving cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). Inducible genes in the bovine placenta/endometrium during early gestation were also characterized by comparing the caruncular (integrated) and intercaruncular (non-integrated) endometrium of pregnancy with endometrium from day 13 of the estrous cycle. A total of 77 genes including 12 unknown genes were induced and about 750 genes including 399 unknown genes were repressed in the caruncular area on day 60 of gestation. While a recent study in humans found that 156 genes were induced and 377 repressed during the implantation window (Kao et al., 2002). The most remarkable change in the present study was found in the PAGfamily, as expression of 16 of the reported 21 genes in cattle (Xie et al., 1997; Green et al., 2000) increased over twofold in the caruncular component but not in the intercaruncular area on day 60 of gestation (Table 3). Other pregnancy-specific genes identified were of the prolactin family, PLs (Nakano et al., 2001) and PRPs (Schuler et al., 1991). Some of these increased up to about fivefold only in the caruncular area. The localization of these peptides in the placentome is in agreement with previous reports (Kessler and Schuler, 1997; Xie et al., 1997; Green et al., 2000; Nakano et al., 2001) and indicates that these genes may have significant role(s) in the adaptation of the endometrium and in placentogenesis in cattle. The high level of expression of PL and PAG family members in early gestation validates their association with trophoblast cell differentiation and indicates that these genes may be pivotal for implantation in this species. Conversely, the expression of uterine serpin (U-serpin), GRP and STC genes was higher in the intercaruncular compared to caruncular area, especially U-serpin which was markedly increased during this period. A much smaller increment was also found in the caruncular sample but this could have arisen from contamination with intercaruncular tissue, as there is no discrete border separating the two areas on the endometrium. Although Stewart et al. (2000) mentioned that U-serpin is a crucial factor for implantation because the lack of this protein causes infertility and it is involved in the synthesis of glandular epithelium, it is indefinite and further studies are necessary. STC-1 has 14 H. ISHIWATA ET AL. TABLE 4. Genes Down-Regulated During Early Gestation in the Bovine Endometrium/Placentome Ratio (pregnancy/estrous) Accession no. Gene name Caruncle Intercaruncle Category no. S54973 AI961832 M59755 NM_006393 AW466068 NM_004428 AW430056 D30750 AW312796 AF162506 AW337037 NM_002940 Bovine 20 alpha-hydroxysteroid dehydrogenase Homo sapiens cDNA clone IMAGE:2512479 30 Bovine llens aldose reductase Homo sapiens nebulette Bos taurus cDNA clone BP230021B20B12 50 Homo sapiens ephrin-A1 Bos taurus cDNA 50 Bovine Msx-1 Bos taurus cDNA 50 Bos gaurus satellite 1.711b Bos taurus cDNA 50 Homo sapiens ATP-binding cassette, sub-family E, member 1 Bos taurus neuronal sodium-dependent glutamate aspartate transporter Bos taurus cDNA clone BP230017A10B11 50 Bos taurus cDNA 50 Human integrin beta 4 Bos taurus matrix Gla protein Homo sapiens fatty-acid-Coenzyme A ligase, long-chain 4 Bos taurus cDNA 50 Bos taurus selp selenoprotein P Homo sapiens cytochrome b5 Bos taurus cDNA clone BP230019A20F7 50 Bos taurus elenoprotein P-like protein Homo sapiens PTD010 Bos taurus ribosomal protein S4 Ovis aries IGF-II B. taurus cathepsin L Homo sapiens peptidase D Bos taurus 11-beta-hydroxysteroid dehydrogenase type 2 Homo sapiens cDNA clone K1599 50 Bos taurus cDNA clone BP230020B20A11 50 Bos taurus cDNA clone BP230013B10F5 50 Homo sapiens actin binding LIM protein Human fetal heart, Lambda ZAP express Homo sapiens cDNA 50 Homo sapiens PHD finger protein 3 Bos taurus clone 2 endothelial adhesion molecule Lu-ECAM-1 Bovine short interspersed repetitive sequences found in genomic DNA Bos taurus futb and rtlf genes Homo sapiens ATP/GTP-binding protein Homo sapiens OS-4 protein Homo sapiens transforming, acidic coiled-coil containing protein 1 Artificial sequences DNA for ART 2 consensus Bovine endozepine Homo sapiens cDNA clone IMAGE:1931520 30 Bos taurus cDNA 50 Bos taurus aquaporin-4-B Ovis aries cyp19 gene Bos indicus ruminant repetitive DNA polymorphism Bos taurus cDNA 50 Bos taurus DNA for SINE sequence Bov-1D Novel human chromosome 1, which has similarities to BAT2 genes Bos taurus DNA for SINE sequence Bov-tA Homo sapiens full length insert cDNA clone ZD18G05 Human pre-mRNA splicing factor SF2p32 Ovis aries prion protein 0.08  0.02 0.09  0.03 0.10  0.04 0.11  0.05 0.12  0.05 0.13  0.04 0.13  0.03 0.14  0.08 0.15  0.05 0.16  0.03 0.16  0.08 0.16  0.05 0.33  0.15 0.39  0.17 0.27  0.15 0.37  0.12 0.37  0.08 0.63  0.34 0.51  0.23 0.38  0.18 0.47  0.16 0.39  0.12 0.41  0.17 0.29  0.12 2 6 2 1 6 2 6 5 6 5 6 3 0.16  0.10 0.62  0.40 2 0.16  0.05 0.16  0.05 0.16  0.02 0.17  0.08 0.17  0.07 0.47  0.18 0.58  0.31 0.49  0.21 0.55  0.16 0.51  0.26 6 6 2 1 3 0.17  0.08 0.17  0.06 0.17  0.08 0.17  0.05 0.18  0.06 0.18  0.06 0.18  0.04 0.18  0.05 0.18  0.06 0.18  0.05 0.19  0.02 0.32  0.14 0.67  0.35 0.42  0.16 0.60  0.31 0.57  0.27 0.45  0.15 0.45  0.11 0.47  0.15 1.01  0.58 0.53  0.21 0.63  0.29 6 2 3 6 2 6 3 2 2 3 2 0.19  0.04 0.19  0.05 0.19  0.03 0.19  0.06 0.19  0.04 0.54  0.19 0.47  0.17 0.51  0.15 0.59  0.26 0.47  0.11 6 6 6 1 6 0.19  0.03 0.19  0.04 0.46  0.14 0.45  0.10 5 2 0.19  0.06 0.48  0.12 6 0.19  0.04 0.19  0.02 0.19  0.06 0.19  0.06 0.45  0.09 0.45  0.09 0.43  0.09 0.61  0.27 3 3 3 6 0.19  0.04 0.20  0.08 0.20  0.04 0.20  0.04 0.20  0.06 0.20  0.02 0.20  0.03 0.49  0.11 0.50  0.17 0.56  0.18 0.54  0.18 0.45  0.10 0.48  0.10 0.45  0.12 6 2 6 6 1 3 6 0.20  0.07 0.20  0.04 0.20  0.07 0.65  0.36 0.57  0.21 0.62  0.27 6 6 6 0.20  0.04 0.20  0.05 0.52  0.16 0.52  0.17 6 6 0.21  0.02 0.21  0.04 0.45  0.09 0.51  0.15 5 1 D82056 AW464852 AW478024 X53587 AF210379 NM_004458 AW484109 AB032826 AB009282 AW465538 D88033 AF078863 D50107 Y16533 X91755 NM_000285 AF074706 N88113 AW465818 AW463728 NM_006719 AA092643 AF091622 AF001262 M26330 AJ132772 NM_006831 AF000152 NM_006283 X82879 M15886 A1350705 AW447717 AB028642 AJ012143 L14584 AW417927 X64126 AL096857 X64124 AF088038 M69039 U67922 (Continued) GENE EXPRESSION PROFILES IN EARLY BOVINE PREGNANCY 15 TABLE 4. (Continued ) Ratio (pregnancy/estrous) Accession no. Z14137 Z93399 U02892 M37210 X53553 S72871 AF027200 AF009329 AJ000518 AF160877 M96455 U25810 D90069 NM_001241 AJ131693 D13891 NM_007011 U52077 J05391 X62882 NM_001930 AF005497 M22559 AF166124 AF191218 X56649 NM_002901 U15731 X56933 AF105429 X52104 AF161464 M18767 AF033096 Gene name Caruncle Intercaruncle Category no. Bos taurus lymphotoxin and tumor necrosis factor alpha Bos taurus lactoferrin Bos taurus Angus 70 kDa heat shock protein-2 Bovine interleukin 1-alpha Bovine IGF-II Human GATA-2 transcription factor Bos taurus rod outer segment guanylate cyclase precursor Rattus norvegicus enhancer-of-split and hairy-related protein 1 Bos taurus BS gene Cercopithecus aethiops intermediate compartment lectin CV1 ERGIC-53 Bos taurus unprocessed alpha-lactalbumin Bos taurus lysozyme Bos taurus adenylate kinase isozyme 2 Homo sapiens cyclin T2 Homo sapiens AKAP450 protein Human Id-2H Homo sapiens putative transmembrane protein Human mariner 1 transposase Bovine glycoprotein III Bos taurus LECAM-1 Homo sapiens deoxyhypusine synthase Bos taurus butyrophilin Bovine ATP synthase inhibitor protein Homo sapiens selenoprotein X Homo sapiens isovaleryl dehydrogenase Bos taurus Annexin I Homo sapiens reticulocalbin 1, EF-hand calcium binding domain Bos taurus somatotropin receptor gene Bos taurus alternative polyadenylation signals Ovis aries H19 Human mRNA for p68 protein Homo sapiens HSPC115 Human complement subcomponent C1s, alpha- and beta-chains Avena sativa nonphototropic hypocotyl 1 0.21  0.09 0.49  0.14 4 0.21  0.07 0.21  0.03 0.21  0.06 0.21  0.09 0.21  0.04 0.21  0.06 0.49  0.12 0.42  0.08 0.49  0.15 0.47  0.15 0.61  0.21 0.46  0.08 2 3 2 2 5 3 0.22  0.06 0.70  0.47 5 0.22  0.02 0.22  0.06 0.52  0.13 0.51  0.22 3 2 0.22  0.07 0.22  0.04 0.22  0.06 0.23  0.11 0.23  0.05 0.23  0.04 0.23  0.03 0.23  0.07 0.23  0.07 0.23  0.10 0.23  0.07 0.24  0.13 0.24  0.04 0.24  0.02 0.24  0.05 0.24  0.07 0.24  0.08 0.46  0.11 0.52  0.22 0.45  0.09 0.80  0.53 0.85  0.51 0.68  0.24 0.40  0.08 0.57  0.19 0.86  0.40 0.47  0.17 0.57  0.23 0.47  0.12 0.47  0.13 0.56  0.17 0.72  0.29 0.51  0.15 0.39  0.20 2 3 3 4 3 4 1 5 2 2 3 2 3 2 3 5 3 0.24  0.07 0.25  0.04 0.25  0.07 0.25  0.10 0.25  0.09 0.25  0.07 0.56  0.30 0.70  0.30 0.65  0.31 0.67  0.22 0.37  0.11 0.74  0.35 2 5 2 5 2 1 0.25  0.08 0.65  0.39 3 Ninety-two down-regulated genes from a total of about 750 annotated genes whose differential expression values were <0.25 between estrous cycle and gestation are shown. Values are meanSD (n¼4). Analysis criteria are shown in Table 3. recently been described in mammals (Chang et al., 1995; Olsen et al., 1996), and is reported to play some role(s) in oocyte maturation and endometrial receptivity during gestation in mice and humans (Harminder et al., 2000). STC-1 expression was found to be particularly high in the intercaruncular area on day 60 of gestation in the present study, indicating a plausible role for this protein in cattle, but this still needs confirmation. The presence of GRP, which is homologous to bombesin and a peptide of the digestive duct, was recently confirmed in the bovine placenta (Jian et al., 1999; Whitley et al., 2000; Budipitoj et al., 2001). In the gastrointestinal tract it contributes to the development of the villi but whether it plays a similar role at the fetal and maternal placental interface needs to be established. The expression of certain genes, such as 20a-hydroxysteroid dehydrogenase (20a-HSD), lens aldose reductase pseudogene, Msx-1, matrix-Gla protein and insulin-like growth factor-II (IGF-II) was decreased on day 60 of gestation in both caruncular and intercaruncular areas (Table 4). In contrast, calbindin gene expression was decreased only in the intercaruncular area (pregnancy/ estrous expression ratio: 0.37  0.09) and not in the caruncular region (0.73  0.11). However, the precise roles of most of these genes during gestation remain unknown (Nikitenko et al., 1998). On the other hand, a decrease of 20a-HSD and IGF-II expression is typical at this stage as progesterone is indispensable for the establishment and maintenance of pregnancy in many species (Kimmins and MacLaren, 2001; Thatcher et al., 2001) and serum IGF-II levels are inversely related to the increasing IGF binding protein concentrations during gestation in cattle (Rogers et al., 1996; Keller et al., 1998). The category of pregnancy-related proteins contained various trophectodermal- and endometrial-specific molecules (Table 5): PAGs, PLs, PRPs, 16 H. ISHIWATA ET AL. TABLE 5. Expression Levels of Selected Pregnancy-Specific Genes in Cattle Annotated genes were selected from those reported in the literature and Gene Database as representative of pregnancy-specific genes. The signal intensity of each gene was estimated by GenePix Pro3.0 software. The classification is based on the expression level of each gene standardized against GAPDH. END, endometrium of day 13 of estrous cycle; ICAR (intercaruncle), CAR (caruncle) and COT (cotyledon) of day 56–64 of gestation. calbindin, U-serpin, GRP and others, as these may be specifically related to the events of implantation and placentogenesis. The accuracy of the results from the array for some of these genes, namely, PAG-1, PL, PRP-1, GRP, calbindin, and STC-1 was independently confirmed by RT-PCR (Fig. 1 and Table 1). Based on their differential expression profiles, the present cDNA microarray profiling strongly suggests that at least 40 genes can be categorized in this species as either pregnancy-related or trophectodermal-and endometrialspecific (Table 5). It is plausible that they are specifically related to the events of implantation and placentogenesis in cattle. This novel analytic technology will yield new insights into trophoblast differentiation and the dynamics of implantation, as mentioned in previous reports (Bilban et al., 2000; Hemberger et al., 2001). GENE EXPRESSION PROFILES IN EARLY BOVINE PREGNANCY 17 Fig. 1. RT-PCR analysis of selected genes. The individual genes that are expressed in bovine caruncular and intercaruncular areas were analyzed by RT-PCR. E, endometrium of day 13 of estrous cycle; 64, CAR and ICAR of day 64 of gestation. PAG-1, pregnancy-associated glycoprotein-1; PL, placental lactogen; PRP-1, prolactin related protein-1; GRP, gastrin-releasing peptide; CaBP, vitamin D-dependent calcium binding protein (Calbindin); STC-1, Stanniocalcin-1. To the best of our knowledge this is the first study to investigate the multitude of genes involved in the implantation process and placentogenesis in cattle using a custom-designed microarray. Our results indicate dynamic expression and interaction of genes from different functional categories and implicate a series of genes not previously known to play roles in implantation and placentogenesis in this species. In addition to providing groups of new genes to consider for their role in health and differentiation of the placenta, our results point to a fundamental role for bovine placental-specific genes, PAGs, PLs, and PRPs, in implantation and placentogenesis in cattle. 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