Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 62, 217-226 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Mouse Oviduct-Specific Glycoprotein Gene: Genomic Organization and Structure of the 5'-Flanking Regulatory Region1

Kazuhiro Takahashib, Yutaka Sendaic, Yoichi Matsudad, Hiroyoshi Hoshic, Masahiko Hiroib, and Yoshihiko Araki2,a,e

a Department of Immunology & Parasitology and b Obstetrics & Gynecology, c Yamagata University School of Medicine, Research Institute for the Functional Peptides, Yamagata-City 990-9585, Japan d Chromosome Research Unit, Hokkaido University Faculty of Science, Sapporo 060-0810, Japan e Center for Reproductive Biology Research, Department of Obstetrics & Gynecology, Vanderbilt University Schoolof Medicine, Nashville, Tennessee 37232-2633


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), can directly associate with gametes or with the early embryo in the oviduct. Although the glycoprotein is widely distributed among mammalian species and there is indirect evidence concerning the involvement of the molecule in the fertilization process, its physiological functions are far from completely understood. To understand the fundamental mechanisms that direct gene expression as well as to know the physiological significance of OGP, we have isolated and characterized a mouse OGP gene (mogp-1). The gene was found to span 13.4 kilobases (kb) including 11 exons and 10 introns. The genomic organization of mogp-1 is well conserved compared to the other members of the chitinase family. Two transcription initiation sites were found at positions 18 and 14 upstream from the first ATG codon. Fluorescence in situ hybridization analysis demonstrated that the mogp-1 was located on the R-positive F3 band of mouse chromosome 3. Although the putative promoter region of mogp-1 lacked typical TATA, CAAT, or GC box sequences, the region contained several motif sequences of transcription factor binding sites including 10 half-palindromic estrogen responsive elements (ERE) and an imperfect ERE. Transient transfection experiments demonstrated that promoter activity could be modulated by various sequences within the 2.2 kb of the 5'-flanking region, and that the mogp-1 promoter was transactivated in an estrogen receptor-positive cell line, MCF-7, by the addition of estradiol-17ß (E2). In addition, relevant promoter activity for E2 responsiveness resides within the first 270 base pairs upstream of the mogp-1. These findings should facilitate our understanding of the regulation of OGP gene expression, and they may be helpful for designing experiments to unravel the role of OGP in the process of mammalian fertilization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the process of mammalian fertilization, essential phenomena, such as recognition and binding between the gametes, and early embryonic development occur in the oviduct [1]. Although normal fertilization and preimplantation development are possible in vitro, there is growing evidence that the oviduct is not just a passive conduit for gamete and embryonal transport but rather that it provides a suitable microenvironment for the fertilization process [1,2]. However, many points remain unclear as to the molecular mechanisms by which oviductal factors affect mammalian fertilization.

Several published reports, including our previous studies, have shown that oviduct epithelial cells secrete specific glycoproteins into the lumen of the oviduct in various mammalian species (for review, see [3–5]). These glycoproteins are specifically expressed within the oviduct during the postovulatory phase of the estrous/menstrual cycle, and several investigators indicate that synthesis of the oviduct-specific glycoprotein (OGP) is controlled by ovarian steroids [3–5]. It has been shown that the OGPs are associated with zonae pellucidae and/or vitelline membranes of oviductal eggs and developing embryos, or they are selectively sequestered into the perivitelline space of eggs [3–5]. It has also been reported that OGP is associated with the sperm surface, and it has been suggested that these glycoproteins may be involved in some sperm function(s) including sperm capacitation, acrosome reaction, or binding to the zona pellucida [3–5]. Recent reports on the cloning of OGP cDNAs from various mammalian species [6–14] have shown that these glycoproteins share high amino acid-sequence identity with a protein recently reported to be a member of the chitinase protein family [15]. However, OGP did not exhibit any hydrolyse activity towards the typical chitinase substrate [9,13]. Despite numerous advances, considerable controversy remains regarding the physiological significance of the glycoprotein in the process of fertilization. To date however, several lines of evidence suggest that the association of OGP with the gamete may have a positive influence on fertilization, at least in vitro [3–5].

In the mouse, the coding region of the OGP cDNA (GenBank accession no. D32137) [10] contains 2163 base pairs (bp) translating to 721 amino acids. Based on comparisons with the N-terminal amino acid sequences of purified bovine [7] and hamster [16] OGPs, it was inferred that the derived amino acid sequence contained a signal peptide region of 21 amino acids and a mature mouse OGP (core protein) region of 700 amino acids [10]. When the N-terminal amino acid sequence of the mouse OGP was compared with sequences of OGPs from other mammalian species [7–14], it was found that these molecules were highly conserved (> 70% identical), whereas the identity was low in the C-terminal side [4,5]. The predicted mouse OGP contains a unique seven-residue repeat sequence (21 repeats) in its C-terminal, which is a species-specific region.

Although there are several studies on characterization and the secretion control by hormones of OGP in several species [3–5], little or no information is available from transcriptional studies on the gene structure and its hormonal regulation. In an attempt to understand the fundamental mechanisms that direct gene expression and the physiological significance of the OGP, we isolated and characterized a DNA segment that encodes the mouse OGP gene. In this study, we constructed recombinant plasmids containing the 5'-flanking genomic region linked to the reporter luciferase gene. Analysis of the promoter activity of the chimeric constructs in transiently transfected estrogen receptor (ER)-positive/negative cell lines allowed us to identify the regulatory sequences that are minimally essential for gene expression under estrogen control.


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

Restriction endonucleases and the random primer DNA labeling kit were from Boehringer Mannheim (Indianapolis, IN). A long-acting polymerase chain reaction (PCR) kit (LA PCR Kit ver.2) and RAV-2 reverse transcriptase were purchased from Takara Shuzo Co. Ltd. (Kyoto, Japan). Modifying enzymes were obtained from Toyobo Co. Ltd. (Osaka, Japan). [{alpha}-32P]dCTP, [{gamma}-32P]ATP, and the 7-deaza-dGTP Sequenase T7 DNA polymerase sequencing kit were purchased from Amersham (Buckinghamshire, UK). The genomic library constructed from the mouse embryonic stem cells (TT2) [17] in the phage vector {lambda}FIX II was kindly provided by Dr. Y. Matsui (Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan). All recombinant DNA technology was done according to standard procedures [18].

Genomic Cloning of Mouse OGP Gene

The genomic library was screened with the digoxigenin-labeled mouse OGP cDNA probe (nucleotides 79–456 in the mouse OGP cDNA [10] produced by PCR) using the plaque hybridization technique as described previously [7,10,11]. The genomic DNA was excised from positive clones and amplified by using PCR with T7, T3, and several oligonucleotide primers corresponding to the mouse OGP cDNA [10]. The PCR products were blunted and phosphorylated with the Klenow enzyme and T4 DNA kinase, respectively, or digested with HindIII when PCR was performed using primers containing the HindIII digestive sequence. After digestion and modification, the products were subcloned into the SmaI or HindIII site of pBluescript II SK(-) or KS(-) vectors (Stratagene, La Jolla, CA).

DNA Sequence Analysis

All nucleotide sequences were determined on both strands using fluorescent-labeled primers and the dideoxynucleotide chain termination method [19] according to the manufacturer's protocol (Applied Biosystems Inc., Foster City, CA). The fluorescent-labeled reaction products were analyzed using a DNA sequencer (Model 373A; Applied Biosystems). Data from DNA sequencing were analyzed by computer-aided sequence analysis (GENETYX-MAC program [Version 6.2.0]; Software Development Co., Ltd., Tokyo, Japan).

Primer Extension Analysis

Total RNA from superovulated mouse oviducts were prepared using the acid guanidium thiocyanate-phenol-chloroform extraction method [20]. Oligonucleotide primers (5 µmol) were end-labeled with T4 polynucleotide kinase and [{gamma}-32P]dATP and purified by ethanol precipitation. Primer extension was performed according to the method described by McKnight and Kingsbury [21] with slight modification as follows: the 5'-end-labeled primer (6 x 105 cpm/5 µmol) was preincubated with 10 µg of the total mouse oviductal RNA in the hybridization mixture (20 µl) containing 250 mM KCl, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA at 60°C for 60 min, and then hybridization was performed at room temperature for 90 min. The hybridization mixture was incubated with RAV-2 reverse transcriptase (20 U) and dNTPs (0.25 mM each) in reaction mixture (50 µl) containing 50 mM Tris-HCl (pH 8.2), 75 mM KCl, 10 mM MgCl2, and 1 mM dithiothreitol at 42°C for 60 min for the primer extension. After reaction, the product was ethanol precipitated and then subjected to a 6% polyacrylamide/8 M urea sequencing gel electrophoresis in parallel with the products of a double-stranded sequencing reaction of M13mp18 as a size marker. The reaction product was visualized using autoradiography.

Chromosomal Localization of Mouse OGP Gene

The direct R-banding fluorescence in situ hybridization (FISH) method was used for chromosomal assignment of the mogp-1 gene (see below). Preparation of R-banded chromosomes and FISH were performed as described previously [22,23]. Briefly, a mitogen-stimulated mouse spleen lymphocyte culture was synchronized by thymidine block, and 5-bromodeoxyuridine was incorporated during the late replication stage for differential replication staining after release of excessive thymidine. R-band staining was performed by exposing chromosome slides to ultra violet light after they were stained with Hoechst 33258. The chromosome slides were hardened at 65°C for 2 h, denatured at 70°C in 70% formamide in double-strength saline sodium citrate (SSC; single-strength SSC is 0.15 M NaCl and 0.015 M sodium citrate), and dehydrated in a 70/85/100% ethanol series at 4°C. The 2.5-kilobase (kb) mouse OGP cDNA fragment inserted into pBluescript II SK(-) [10] was labeled by nick translation with biotin-16-dNTP (Boehringer Mannheim) according to the manufacturer's protocol. The labeled DNA fragment was ethanol-precipitated with salmon sperm DNA and Escherichia coli tRNA, and then denatured at 75°C for 10 min in 100% formamide. The denatured probe was mixed with an equal volume of hybridization solution to a final concentration of 50% formamide, double-strength SSC, 10% dextran sulfate, and 2 mg/ml BSA. Twenty microliters of the mixture, containing 250 ng labeled DNA, was put on the pretreated slides, covered with parafilm, and incubated overnight at 37°C. The slides were washed for 20 min in 50% formamide in double-strength SSC at 37°C, and then without formamide in double-strength SSC and in single-strength SSC for 20 min each at room temperature. After a final rinse in 4-strength SSC, they were incubated under coverslips with antibiotin antibody (Vector Laboratories, Burlingame, CA) at a 1:500 dilution in 1% BSA/4-strength SSC for 1 h at 37°C. They were then washed with carbocyanine 2-labeled donkey anti-goat IgG (Amersham) at a 1:500 dilution for 1 h at 37°C. After being wash sequentially with 4-strength SSC, 0.1% Nonidet P-40 in 4-strength SSC, and 4-strength SSC for 10 min each on the shaker, the slides were rinsed with double-strength SSC and stained with 0.75 µg/ml propidium iodide. Excitation at wave lengths 450–490 nm and near 365 nm were used for observation (filter sets B-2A and UV-2A, respectively; Nikon, Tokyo, Japan).

Construction of Chimeric Luciferase Plasmid

Chimeric plasmids were constructed by subcloning from various portions of the 5'-flanking region of the mogp-1 gene (see below) into a reporter vector, PGV-Basic, containing the luciferase gene but no eukaryotic regulatory elements (Toyo Ink Mfg., Tokyo, Japan). PCR was performed to generate the Luc-HD chimeric plasmid using a primer pair: 5'-AGAGCCAAAGCTTTGAAACT-3', corresponding to -373 to -354 of the mogp-1 sense sequence (see below), and 5'-GGGAAGCTTCTCAACTGCCTGGTAGCTCTGG-3', corresponding to -4 to 18 of the mogp-1 antisense sequence (see below) plus the HindIII digestive site at the 5' end of the primer (underlined). pMG1, a plasmid clone used for the production of chimeric clones, was generated as follows: The 14.8-kb NotI fragment from {lambda}mg1 (a genomic phage clone containing part of mogp-1; see below) was subcloned into the NotI site of pBluescript II KS(+) vector and then used for PCR amplification. The PCR product (401 bp) was digested with HindIII, and the digested 385-bp-length fragment was introduced into the HindIII site of PGV-Basic to obtain Luc-HD. The 1908-bp fragment of the 5'-flanking region of the mogp-1 gene was obtained by cutting the pMG1 with BamHI (internal site: -2216) and AvrII (internal site: -308). Luc-BM was constructed by inserting the 1908-bp fragment into the site from which the BamHI (polylinker site)/AvrII (internal site) fragment of the Luc-HD had been removed. Luc-BM was completely digested with KpnI (polylinker site/internal site: -1060), and religated to generate Luc-KN. Luc-SP was obtained by complete digestion of the Luc-BM with SmaI (polylinker site) and PvuII (internal site: -143) and religating the chimeric plasmid on itself. Luc-XP was generated by digesting the Luc-BM with XhoI (polylinker site) and PstI (internal site: -48), blunting with the Klenow enzyme, and religating the chimeric plasmid on itself. A vector (PGV-C), constructed by introducing the SV40 promoter into the PGV-Basic vector, was used as a positive control for assaying promoter activity. Propagation of all plasmids was done in the bacterial strain DH5{alpha} (Toyobo) using 50 µg/ml ampicillin in LB medium. Plasmid DNA for transfection was purified using the QIAGEN plasmid Midi kit (Qiagen, Hilden, Germany).

Cell Culture and Transient Transfection

Cryopreserved MCF-7, an ER-positive human breast cancer cell line, and a Chinese hamster ovary cell line (CHO-K1) that has been reported to be ER-negative [24] were provided from the Cancer Cell Repository, Tohoku University, Japan. They were maintained in RPMI-1640 medium (ICN Biochemicals Inc., Aurora, OH) or Dulbecco's modification of Eagle's medium (DMEM) supplemented with Ham's F-12 nutrient mixture (1:1) (Life Technologies, Inc., Gaithersburg, MD), which were both supplemented with 10% newborn calf serum (Mitsubishi Chemical Industries Ltd., Tokyo, Japan), at 37°C in 5% CO2. Transfection efficiencies were monitored by use of a pSV-ß-galactosidase vector containing E. coli lacZ gene under the SV40 promoter (Promega, Madison, WI). Cells at an exponential growth phase (1–4 x 105 cells) were seeded into 6-well plates or 35-mm culture dishes. When those cells reached approximately 80% confluence, the culture medium was changed to phenol red-free RPMI-1640 or DMEM/F-12, which both contained 5% charcoal-dextran-treated fetal calf serum (stripped serum), and maintained for 24 h. The cells were co-transfected using the calcium phosphate method [18] with 4 µg of chimeric plasmid and 2 µg of pSV-ß-galactosidase vector. Four hours after transfection, the cells were washed once with PBS (pH 7.4) and treated with 15% (w:v) glycerol/Hebs (21 mM HEPES, 145 mM NaCl, 0.7 mM Na2HPO4) for 30 sec at room temperature, and then rinsed twice with PBS. Next the cells were incubated with new culture medium (phenol red-free RPMI-1640 or DMEM/F-12, both containing 5% stripped serum) with or without 10-7 M estradiol-17ß (E2) for 48 h at 37°C in 5% CO2 in air. After incubation, the cells were washed with PBS and then dissolved in PicaGene cell lysis buffer LCß/PGC-51 (Toyo Ink Mfg.). The luciferase reaction was performed at room temperature by mixing 20 µl of cell lysate with 100 µl of luciferase substrate solution containing 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 20 mM N-Tris(hydroxymethyl)methylglycine, 1.07 mM (MgCO3)4Mg(OH)2·5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 mM coenzyme A, 470 mM luciferin, and 530 mM ATP. Immediately after mixing, the light intensities emitted from the samples were measured on a Luminescencer-JNR (ATTO, Tokyo, Japan). To normalize transfection efficiency, ß-galactosidase assay was performed on each sample according to the standard procedure [18]. Incubation with o-nitrophenyl-ß-D-galactopyranoside produced a yellow stain that was measured on a Beckman (Palo Alto, CA) Du 640 spectrophotometer at 420 nm. Statistical analysis was performed using Student's t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Preliminary Characterization of Mouse OGP Gene

To isolate the mouse OGP gene, a probe representing the mouse OGP open reading frame [10] was used to screen a genomic library prepared from the mouse TT2 embryonic stem cell line. From an initial screening, 4 independent clones were isolated and characterized further. Of these clones, two ({lambda}mg1 and {lambda}mg4) were chosen for further analysis since they overlapped in part and covered approximately 16 kb of the gene. The positions of the phage clones in relation to the mouse OGP gene are shown schematically in Figure 1. Nucleotide sequence analysis revealed that those clones covered 16 166 bp containing all the exons and introns of the mouse OGP gene as shown in Figure 2. The mouse OGP gene, termed mogp-1, was composed of 11 exons and 10 introns, spanning approximately 13.4 kb from exon 1 to exon 11. The species-specific coding region at the C-terminus [4,5] was located en bloc in exon 11. The exon-intron splice sites were confirmed by the GT/AG rule [25] (Table 1). The first exon was 43-bp long, and an 18-bp 5'-untranslated region preceded the first ATG that encodes the initiation methionine. The sequence encoding signal peptide spanned from exon 1 to exon 3 (Fig. 2).



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  		FIG. 1. Structure of mogp-1. A) Two overlapping genomic clones containing the mouse OGP locus, and partial restriction maps of {lambda}mg1 and {lambda}mg4. The clones are aligned to show the 13.0-kb region overlap. Restriction endonucleases used: B, BamHI; H, HindIII; X, XbaI; K, KpnI; E, EcoRI. B) Exons (exons 1–11) are indicated by the solid boxes. Introns are lettered A–J. At the top of the figure is the kilobase scale marker



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  		FIG. 2. Sequence of mogp-1. The exons are boxed and the protein coding sequence shows triplet codons. The farthest transcriptional initiation site is assigned as nucleotide 1 at residue A, and the sequential nucleotide number of the mouse OGP cDNA is shown on the top and bottom of each exon. Uppercase and lowercase letters indicate exons and introns including the 5'- and 3'-flanking region, respectively. Italic capital letters indicate the putative signal peptide sequence of mogp-1. Poly(A) signal is underlined, and asterisks indicate the stop codon of the mouse OGP cDNA


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  		TABLE 1. Exon/intron boundaries of mogp-1.*

Identification of the Transcription Start Site

To determine the transcription initiation site(s), primer extensions were performed using total RNA isolated from mouse oviduct at the postovulational phase. The 24-mer oligonucleotide primer (5'-TGGTGAAATAGCACACCAGTTTGT-3', corresponding to mouse OGP79–102 antisense cDNA sequence [10]), gave two initiation sites (Fig. 3). In an additional confirmatory primer extension analysis using 5'-GCACACCAGTTTGTAGGCAGTACC-3', corresponding to mouse OGP69–92 antisense cDNA sequence [10], a similar band pattern was observed (data not shown). When the putative transcription start sites determined by primer extension were compared to the mouse OGP cDNA sequence [10], it was found that results obtained from these experiments agree in terms of the length of the 5' untranslated region. Since the intensity of two extending products obtained by primer extension were almost identical (as shown in Figure 3), these data suggest that the start sites were localized at nucleotides 18 and 14 upstream from the first ATG initiation codon. The most upstream site was designated as nucleotide +1 as shown in Figure 4.



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  		FIG. 3. Identification of transcription initiation sites in mogp-1. An oligonucleotide antisense primer, corresponding to mouse OGP79–102 cDNA sequence [10], was hybridized with 10 µg of total RNA isolated from the mouse oviduct (lane 1), or without total RNA (lane 2). The extension products were analyzed using an 8 M urea/6% acrylamide sequencing gel as described in Materials and Methods. M13mp18 was used as a size marker. The arrows indicate the positions corresponding to the extended products with a length of 102 and 106 nt, respectively



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  		FIG. 4. Nucleotide sequence and putative regulatory elements of the 5'-flanking region of mogp-1. The encoding sequence of the first exon is indicated by a box; two transcription start sites are indicated by asterisks, and the most upstream start site is numbered as +1 (on far left of box). The major restriction enzyme sites used for making chimeric plasmid are indicated by double underlines. The sequence of 5'-half ERE, as well as 3'-half ERE are highlighted in gray shadow, and imperfect ERE is indicated by black box. Consensus sequences for second B cell-specific octamer binding protein (OTF-2B) [53,54], leader-binding protein-1 (LBP-1) [55], VP-16 [56], TGGCA [57], cAMP response element (CRE) [58], CACCC-binding [59,60], T-antigen [61], erythroid-cell-specific nuclear factor (NF-E1) [62–64], TATA-like sequence (TATTAA) [29], and CAAT-like sequence (CAAC) are underlined. Lowercase letters represent the partial nucleotide of the first intron. Numbers along side of the sequence refer to nucleotide position relative to the transcription start site

Sequencing and Analysis of the 5'-Flanking Region of mogp-1

Although the putative promoter region of the mogp-1 gene lacked the typical sequence of TATA, CAAT, and GC boxes [26–28], TATA-like (TATTAA) [29] and CAAT-like (CAAC) nucleotide sequences were identified at positions -29 and -36, respectively (Fig. 4). Computer-aided analysis of the 5'-flanking region of the gene revealed that there are several consensus sequences for binding of transcription factors as shown in Figure 4. In particular, 5'- or 3'-half-palindromic estrogen responsive elements (ERE) [30–32] were found in the 5'-flanking region (5'-GGTCA-3' at -2199, -1683, -1661, -1533, -1312, -1211, -728, -174; 5'-TGACC-3' at -2167, -974; as well as an imperfect ERE [33] at -109) (Fig. 4).

Chromosomal Localization of mogp-1

The chromosomal location of the mogp-1 gene was determined by the direct R-banding FISH method using mouse OGP cDNA as a probe. The mogp-1 gene was localized to the R-positive F3 band of chromosome 3 (Fig. 5) [22,34,35], where a conserved linkage homology to human chromosome 1 has been identified [36].



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  		FIG. 5. Chromosomal mapping of mogp-1: chromosomal localization of mogp-1 on the mouse R-banded chromosome using a cDNA fragment as a biotinylated probe. The hybridization signal is indicated by the arrow. The signals are localized in the R-positive F3 band of mouse chromosome 3. The metaphase spreads were photographed with Nikon B-2A (a) and UV-2A (b) filters. R- and G-banded patterns are demonstrated in a and b, respectively

Analysis of the Functional Activity of mogp-1

A promoter fragment containing various lengths of the 5'-flanking sequence of mogp-1, ending within the untranslated region of the first exon, was fused to a luciferase reporter gene to obtain the deletion clones Luc-BM, Luc-KN, Luc-HD, Luc-SP, and Luc-XP (Fig. 6). To determine the estrogen inducibility of mogp-1 gene expression, each chimeric plasmid was transfected into both MCF-7 (ER-positive) and CHO-K1 (ER-negative) cells with or without E2 in the culture medium. Activity of the Luc-BM, Luc-KN, and Luc-HD constructs were significantly (P < 0.05) induced (21.8 ± 5.4, 12.6 ± 4.8, and 7.3 ± 2.1-fold, respectively; means of three independent experiments) by the addition of 10-7 M E2 in the transfected MCF-7 cells (Fig. 7). The Luc-SP construct, containing an imperfect ERE (5'-GGTCATTGTGACT-3') at the position of -109, was also inducible. However, the deletion of an additional 127 bp containing the 5'-half ERE from Luc-HD significantly decreased estrogen inducibility as shown by the luciferase expression observed for the Luc-SP construct (2.5 ± 0.8-fold). No estrogen inducibility was found for the ERE-negative luciferase expression construct, Luc-XP. As shown in Figure 7B, no constructs transfected in the ER-negative CHO-K1 cell line conferred stimulation of luciferase activity by the addition of E2.



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  		FIG. 6. Structure of chimeric plasmid. A) Partial restriction map of 5'-flanking region of the mogp-1. Arrows indicate the transcription start site (+1) and initiation codon (ATG). Hatched and black boxes indicate, respectively, the untranslated region and the transcribed region of exon 1 of the mogp-1. B) The coding region of luciferase gene was fused to fragments of the 5'-flanking region of mogp-1, all ending within the first exon, to generate a series of deletions of the Luc-chimeric plasmids. Open triangles, black triangles, and black diamonds indicate, respectively, 5'-half EREs, 3'-half EREs, and imperfect EREs. The 5'-flanking sequence of the mogp-1 contained in each construct is numbered relative to the transcription start site. The mogp-1 gene and the luciferase coding region are represented by filled boxes and open boxes, respectively



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  		FIG. 7. E2-induced luciferase reporter gene activity of mogp-1. Various mogp-1 promoter-luciferase reporter constructs were transiently transfected into MCF-7 cells (A) and CHO-K1 cells (B). Cells were cultured in media with 10% stripped fetal calf serum for 24 h and then transfected with chimeric plasmid (4 µg) and pSV-ß-galactosidase (2 µg). At 4 h after transfection, E2 (10-7 M) in ethanol or ethanol alone was added to the medium. Transfection efficiency was normalized to the ß-galactosidase activity, and the luciferase activity is expressed as fold increase over that of the Luc-BM without E2 induction. Numbers in parentheses indicate the fold induction. Data are the mean ± SE from three different transfection experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we isolated and characterized a DNA fragment encoding the mouse OGP gene, termed mogp-1. The mogp-1 gene spans 13.4 kb, represented as eleven exons separated by ten introns (Fig. 2). Results from nucleotide sequence analysis revealed that there was no sequence discrepancy between the genomic DNA and the mouse OGP cDNA as previously reported [10]. Exon/intron boundaries of the gene conformed to known consensus splice donor and acceptor motifs [25]. Although mogp-1 possessed a TATA-like sequence (TATTAA [32] at the position of -29 in the 5'-flanking region), no canonical TATA/GC box sequence was observed in the putative promoter region. It has been reported that the genes lacking a TATA box fall into two groups [37]. One class of the genes has a GC-rich promoter. Usually, this class of genes contains several transcription initiation sites and potential binding sites for the transcription factor Sp1, whereas the other class has no GC-rich region in the 5'-flanking region. Typical genes belonging to the latter class that have been reported include the Drosophila homeotic gene [38,39] and acrosin (a sperm acrosomal serine protease) gene [40]. Sequence analysis of the 5'-flanking region of mogp-1 demonstrates that this gene also appears to belong to the latter class of genes (Fig. 4). A CAAT-like sequence (CAAC) was found at -36 of mogp-1; however, the CAAC may not be functional since a functional CAAT sequence is generally conserved at the position of -77 ± 10 region from the mRNA capping site [27].

Comparison of the structural organization of mogp-1 to that of other members of the chitinase family demonstrates their similarities, as is shown in Figure 8. All chitinase family members—such as the human chitotriosidase [41,42], human cartilage glycoprotein-39 (HC gp-39; GenBank accession no. Y08374–Y08378) [15,43], and human OGP (a homologue to mouse OGP; U58001–U58010) genes—are composed of 10 or 11 exons, and the pattern of intron insertion is also highly conserved. It should be noted that the number and position of cysteine residues are highly conserved among the proteins encoded by the genes (Fig. 8). In addition, these similarities concerning the cysteine residues are also observed in the cDNA of 39-kDa human chondrocyte protein with the N terminus YKL (YKL-39; U49835) [44] and mouse macrophage secretory protein (YM-1; M945849), which were recently identified as members of the chitinase family, suggesting that these proteins arose from a common ancestral gene and may have evolved by gene duplication.



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  		FIG. 8. Comparison of exon structure of mogp-1 with those other chitinase family proteins. Exon in mogp-1, human OGP gene, human chitotriosidase gene, and HC gp-39 gene, and cDNAs of YKL-39 and YM-1 cDNA are indicated. The open boxes represent the coding region of the exons, while the 5'- and 3'-untranslated regions are indicated by the black boxes. The phase class of introns are shown as 0 (intron occurs between codons), I (intron interrupts the first and second bases of codon), and II (intron interrupts the second and third bases of codon). The hatched boxes represent the coding region of the signal sequence. The arrows indicate the translation initiation site (ATG), and the inverted, closed triangles represent conserved cysteine residues

Data obtained by FISH analysis demonstrated that mogp-1 was located on the R-positive F3 band of mouse chromosome 3. Previous studies have reported that both RAP1A, a member of human RAS oncogene family, and KCNA, a voltage-gated potassium channel gene, were located on human chromosome 1 (1p12–13 and 1p13.3, respectively) [45,46] in the vicinity of human OGP gene (1p13), whereas Rap1a [47] and Kcna3 [48], mouse genes homologous to the human genes, were identified on mouse chromosome 3, suggesting that this area of the mouse chromosome 3 is a syntenic region to human chromosome 1. The present study revealed that the mogp-1 gene was also located in the vicinity of Rap1/Kcna3 genes. These results should support that the R-positive F3 band of mouse chromosome 3 shows conserved linkage homology to human chromosome 1 [49].

Sequence analysis of the 5'-flanking region of mogp-1 revealed that it contains various putative regulatory elements, providing insights into the pattern of regulation of mogp-1 gene expression. Of these elements, it is well known that the imperfect ERE as well as the half-palindromic ERE have been shown to play a role in the regulation of estrogen-dependent gene expression [33]. The classical consensus ERE (GGTCANNNTGACC) was originally defined from the Xenopus vitellogenin A2 gene promoter, indicating that ER binds directly to this region [50]. The estrogen-responsive finger protein gene also utilizes a consensus ERE for its regulation [51]. To date, however, it has been reported that most estrogen-responsive genes contain one or more imperfect ERE or multiple copies of the ERE half-site rather than the consensus ERE [31]. Previous studies including our preliminary studies have suggested that mammalian OGP is secreted from oviductal epithelial cells in an E2-dependent manner, although some parts of the regulation still remain controversial (for review see [2–5]). To identify the transcriptional region of mogp-1 gene expression controlled by estrogen, we then attempted to perform transient transfection studies, using chimeric plasmid constructions (Fig. 6), into ER-positive/negative cell lines. Transient transfection assays shown in Figure 7 indicate that a 2.2-kb segment of the 5'-flanking region specifically directs the transcription of a reporter gene in an ER-positive cell line, MCF-7, by E2. Moreover, deletion of the 2.2 kb, including the putative transcription initiation site, abolished promoter activity. Results from these functional analyses support transcript mapping and sequence analysis data, suggesting that this DNA segment is the mogp-1 proximal promoter and is controlled by estrogen. Indeed, our preliminary studies, using transgenic mice carrying a heterologous DNA encoding the SV40 T-antigen under the 2.2-kb segment, indicated that estrogen-dependent tumor formation was also observed in these mice (unpublished results).

Transfection of MCF-7 cells with constructs deleting either 1156 bp (Luc-KN) or 1946 bp (Luc-HD) from the 5'-end of the 2.2-kb promoter region resulted in a similar reporter activity. However, an additional 127-bp deletion from the Luc-HD (Luc-SP) showed significant reduction of the reporter gene expression. Although the 143-bp upstream region in Luc-SP contains an imperfect ERE, this motif may be not complete enough for sufficient gene regulation to be induced by E2. A previous report identified an imperfect ERE in the lactoferrin gene promoter that was able to regulate gene expression [33]. It should be noted, however, that the imperfect ERE in the lactoferrin gene shared five nucleotides (GGTCA) with the chicken ovalbumin upstream-promoter transcription factor [33], whereas the imperfect ERE at position -109 of mogp-1 did not overlap any reported transcriptional factor binding motif (Fig. 4). This could be the reason for the lack of response to estrogen stimulation in the Luc-SP transfection. Taken together, these results allow us to conclude that the 2.2-kb segment contains a promoter activity regulated by E2 and that a relevant promoter activity for estrogen responsiveness resides within the first 270 bp upstream of mogp-1. Further studies will be required to identify the actual ER binding site(s) and the motif regulating the tissue specificity necessary for both basal and inducible regulation of mogp-1 gene.

In recent years, considerable progress has been made in the identification and molecular characterization of OGP, which has been widely identified in mammalian species including humans. Although it has been suggested that OGP may be involved in some functions in gametes, zygotes, and/or early embryos, its physiological significance in mammalian fertilization process remains controversial. The findings reported here provide a framework for further analysis of the regulation of mogp-1 expression and OGP structure-function relationships. Such studies will be critical for understanding and eventually modulating the role of OGP during the fertilization process. Now that the structure of mogp-1 has been characterized, we can proceed to in vivo functional analyses, such as loss-of-function assays in mice, by targeted gene disruption. Since germ-line competent TT2 embryonic stem cells lacking mogp-1 have been already established [52], we are assessing the fertilization process in homozygous mice.


   ACKNOWLEDGMENTS
 
 The authors are indebted to Dr. Yasuhisa Matsui (Osaka Medical Center and Research Institute for Maternal and Child Health) for providing the mouse TT2 genomic library used in this study. We gratefully acknowledge Drs. Fujiro Sendo, Hiromi Yoshida-Komiya (Yamagata University), Marie-Claire Orgebin-Crist, Daulat Ram P. Tulsiani (Vanderbilt University), Ichiro Miyoshi, and Noriyuki Kasai (Tohoku University) for their helpful discussions and encouragement throughout the course of this study. We are deeply indebted to Ms. Tomoko Onuma, Atsuko Kurita, and the staff of Animal/RI Center, all in Yamagata University, for their technical supports. We are grateful to Dr. Benjamin J. Danzo (Vanderbilt University) for critically reading the manuscript.


   FOOTNOTES
 
First decision: 19 August 1999.

1 This work was supported in part by Grants-in-Aid for General Scientific Research (Nos. 08671862 & 09671663) and for Scientific Research, International Scientific Research Program (No. 07044220) from the Ministry of Education, Science and Culture, Japan; and by a grant from the Ichiro Kanehara Foundation. The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL, and DDBJ data banks with the accession number AB006193. Back

2 Correspondence: Yoshihiko Araki, Center for Reproductive Biology Research, Department of Obstetrics & Gynecology, Vanderbilt University School of Medicine, 1161 21st Avenue South, Room U3305 MCN, Nashville, TN 37232-2633. FAX: 615 343 7797;yoshihiko.araki{at}mcmail.vanderbilt.edu Back

Accepted: September 13, 1999.

Received: July 20, 1999.


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