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Vol. 142, No. 3 Printed in U.S.A.

Gene Duplication Gives Rise to a New 17-Kilodalton Lipocalin That Shows Epididymal Region-Specific Expression and Testicular Factor(s) Regulation* JEAN-JACQUES LAREYRE†, VIRGINIA P. WINFREY, SUSAN KASPER, DAVID E. ONG, ROBERT J. MATUSIK, GARY E. OLSON, AND MARIE-CLAIRE ORGEBIN-CRIST Departments of Obstetrics and Gynecology (J.-J.L., M.-C.O.-C.), Biochemistry (D.E.O.), Cell Biology (V.P.W., S.K., R.J.M., G.E.O., M.-C.O.-C.), and Urologic Surgery (S.K., R.J.M.) and Center for Reproductive Biology Research (S.K., D.E.O., R.J.M., G.E.O., M.-C.O.-C.), Vanderbilt University, Nashville, Tennessee 37232 ABSTRACT Using transgenic mice, we have recently shown that 5 kb of the 5⬘-flanking region of the mouse epididymal retinoic acid-binding protein (mE-RABP) gene contains all of the information required for spatial and temporal gene expression in the epididymis. To identify the important cis-DNA regulatory element(s) involved in the tissue-, region-, and cell-specific expression of the mE-RABP gene, the 5-kb DNA fragment was sequenced. A computer analysis of the nucleotide sequence showed the presence of a new gene located 1.7 kb upstream from the mE-RABP gene transcription initiation site. The analysis of the open reading frame showed that the new gene encoded a putative 17-kDa lipocalin (named mEP17) related to mE-RABP. A 600-bp complementary DNA encoding mEP17 was cloned by rapid amplification of 3⬘-cDNA ends from epididymal total RNA. Two mEP17 RNA species (1 and 3.1 kb in size) were detected by Northern blot in the epididymis, but not in other tissues tested. In situ hybridization anal-

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AMMALIAN SPERMATOZOA undergo a complex process of maturation in the epididymis, resulting in their forward motility and fertilizing ability (1). In the epididymis, spermatozoa are exposed to a microenvironment created by the absorptive and secretory activities of the epithelial cells. It is believed that interaction between epididymal secretory proteins and the sperm plasma membrane is involved in the sperm maturation process. The epididymal function is mainly regulated by androgens (2). However, a vitamin A-free diet or the expression of a dominant negative of the retinoic acid receptor ␣ in the mouse cauda epididymidis (3) results in the degeneration of the epithelium and Received August 17, 2000. Address all correspondence and requests for reprints to: Dr. MarieClaire Orgebin-Crist, Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Medical Center North, Room C-3306, Nashville, Tennessee 37232-2633. E-mail: m-c.orgebin-crist@ mcmail.vanderbilt.edu. * This work was supported by NIH Grants HD-03820, HD-05797, HD-36900, HD-25206, and HD-20419. The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL databank with accession number AF082221. † Present address: Institut National de la Recherche Agronomique, Station Commune de Recherches en Ichtyophysiologie, Biodiversite´ et Environnement, Campus de Beaulieu, 35042 Rennes Cedex, France. E-mail: [email protected].

yses showed that, unlike mE-RABP messenger RNA (mRNA), which is expressed in the distal caput epididymidis, mEP17 mRNA was detected only in the principal cells of the initial segment. The spatial expression and homology with mE-RABP suggest that mEP17 may act as a retinoid carrier protein within the epididymis. mEP17 mRNA expression disappeared 5 days postcastration. Four days after unilateral castration, mEP17 mRNA had nearly disappeared in the epididymis from the castrated side, but not from the intact side. In addition, testosterone replacement to bilaterally castrated mice failed to restore gene expression. We conclude that mEP17 gene expression is dependent on testicular factors circulating in the luminal fluid. Together our results suggest that mE-RABP and mEP17 genes were generated by duplication and that evolution led to a different regionspecific gene expression and regulation in the epididymis. (Endocrinology 142: 1296 –1308, 2001)

male infertility. Therefore, epididymal function is also dependent on the delivery of retinoids. We previously identified a mouse epididymal secretory protein (MEP 10) (4), now named murine epididymal retinoic acid-binding protein (mE-RABP), that is specifically synthesized by principal cells of the mouse mid/distal caput epididymidis and is secreted into the tubule (4). Once in the luminal fluid, mE-RABP is in contact with spermatozoa, but does not bind tightly to them, as it was not detected after washing (4). The deduced amino acid sequence of mE-RABP is highly homologous (75% identity, 90% homology) to two rat epididymal retinoic acid-binding proteins successively named proteins B/C (5, 6), epididymal-binding protein-I and -2 (7), epididymal secretory protein I (ESPI) (8), and E-RABP (9). These proteins are not restricted to rodent epididymis, because the N-terminal sequences of two boar epididymal secretory proteins are highly similar to mE-RABP and ESP-I (10). The mE-RABP and ESP-I proteins belong to the lipocalin superfamily (11, 12). The three-dimensional structure analyses reported to date show that lipocalins have an eight up and down stranded ␤-barrel closed at one end by a single turn of ␣-helix forming a hydrophobic binding cavity. This hydrophobic pocket is well adapted for noncovalent binding and transport of small lipophilic ligands. The mE-RABP binds active retinoids (9-cis and all-trans-retinoic acid) (13)

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and therefore may function as a carrier protein and mediate a retinoic acid signal pathway in the mouse epididymis (14). The gene encoding mE-RABP was isolated and mapped to the locus [A3,B] of mouse chromosome 2 (15). We have recently shown that 5 kb, but not 0.6 kb, of the 5⬘-flanking region of the mE-RABP gene targeted high levels of chloramphenicol acetyl transferase reporter gene expression to the principal cells of the mid/distal caput epididymidis in transgenic mice (16). Transgene expression was consistent with the temporal and spatial expression pattern of the endogenous mE-RABP messenger RNA (mRNA) and was regulated by androgens. This demonstrates that cis-DNA regulatory elements important for epididymis-, region-, and cellspecific gene expression and regulation are located within the 5 kb upstream from the mE-RABP gene. In this study we identified within 5 kb of the 5⬘-flanking region of the mE-RABP gene a new gene encoding a 17-kDa lipocalin. This protein was named mEP17 for mouse epididymal protein of 17 kDa. Northern blot and in situ hybridization analyses were carried out to study the tissue-, region-, and cell-specific expression as well as regulation of the mEP17 mRNA. Further, we demonstrate that a mEP17-like gene is conserved in the rat and hamster genome. The conservation of mEP17-like gene expression during evolution suggests that it may play an important function in male fertility. Materials and Methods Animals All experiments were conducted in accordance with the NIH Guidelines for Care and Use of Animals in the Laboratory. Mice were kept under constant conditions of temperature (20 ⫾ 1 C) and light (12 h/day), with water and food available ad libitum. Organs were obtained from adult B6D2/F1 mice (Harlan Sprague Dawley, Inc., Indianapolis, IN). Castration or efferent duct ligation was performed by the abdominal route under light methoxyflurane (Mallinckrodt, Inc., Mundelein, IL) anesthesia. When required, hormone replacement was carried out with daily sc injection of testosterone propionate (2 ␮g/g), dissolved in sesame oil. Mice were killed 1 day after the last injection, and organs were excised, immediately frozen in liquid nitrogen, and stored at ⫺80 C.

Isolation and sequencing of genomic DNA fragments DNA fragments from the Bac clone 10983, containing 5 kb of the 5⬘-flanking region of the mE-RABP gene (15), were subcloned in pBluescript SK⫹ using appropriate enzymes (Promega Corp., Madison, WI). DNA templates for sequencing were purified using the Plasmid Midi kit (QIAGEN, Santa Clarita, CA). Sequencing reactions were performed as described in the Thermo Sequenase fluorescent-labeled primer cycle sequencing kit (Perkin-Elmer Corp., Foster City, CA). DNA fragments were separated in a denaturing PAGE (6% acrylamide gel) and analyzed using an ABI 373A automated sequencer (PE Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed using the GeneJockey sequence processor (Biosoft, Milltown, NJ).

Primer extension analysis Total RNA was extracted from the mouse epididymis using the method described previously (17). The mEP17PE2 primer (5⬘-CTTCTCTGGTACAAGCTCCACCCTGGT-3⬘) specific for mEP17 mRNA was radiolabeled using T4 polynucleotide kinase in the presence of 100 ␮Ci [␥-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s instructions (New England Biolabs, Inc.). For each reaction, 10 ␮g epididymal total RNA or transfer RNA were hybridized to 1 pmol (105 dpm) mEP17PE2 primer for 12 h at 35 C in 10 ␮l of a solution containing 0.04 m PIPES (pH 6.4), 1 ␮m EDTA, and 80% (vol/vol) formamide. RT was performed in 20 ␮l con-

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taining 50 ␮m Tris-HCl (pH 8.3), 30 ␮m KCl, 8 ␮m MgCl2, 6 ␮m dithiothreitol, 0.5 mm of each deoxy-NTP, and 50 U avian myeloblastosis virus (AMV) reverse transcriptase (Promega Corp.). Samples were incubated for 30 min at 42 C, and then 50 U AMV reverse transcriptase were added again and incubated for 1 h more. Elongated radiolabeled fragments were loaded on denaturing PAGE (7% acrylamide gel) next to sequencing reactions carried out using the Sequenase sequencing kit (Amersham Pharmacia Biotech). The clone pHindIII (15) and the mEP17PE2 primer were used as template and primer, respectively.

Isolation of a complementary DNA (cDNA) encoding mEP17 For RT, 2 ␮g epididymal total RNA were incubated in 50 mm Tris-HCI (pH 8.3); 75 mm KCl; 10 mm dithiothreitol; 3 mm MgCl2; 0.5 mm each of deoxy (d)-GTP, dATP, dCTP, and dTTP; 2 U/␮l RNasin (Promega Corp.); 10 ␮g/ml oligonucleotide RACEIII (5⬘-CAGCTGCAGGTACCGGATCCTCGAGAAGC(T)18-3⬘); and 200 U Moloney murine leukemia virus reverse transcriptase for 1 h at 42 C. The DNA/RNA hybrids were denatured for 5 min at 90 C and stored at ⫺80 C. PCR was performed using 100 ng cDNA incubated in a mixture containing 0.2 mm each of dGTP, dATP, dTTP, and dCTP; 1 ␮m of each primer (FwmEP17cDNA, 5⬘-GGCCCTGAGATGGAAGCTAGG-3⬘; RiIII, 5⬘-GCAGGTACCGGATCCTCGAGAAG-3⬘); 1 ⫻ reaction buffer II (Perkin-Elmer Corp.); and 1 U AmpliTaq (Perkin-Elmer Corp.). DNA was amplified for 29 cycles, consisting of 1 min at 94 C, 2 min at 55 C, and 2 min at 72 C. PCR products were purified on 2% (wt/vol) agarose gels, ligated into pGEM-T easy plasmid (Promega Corp.), and sequenced.

Southern blot analyses of genomic DNA Genomic DNA was extracted from the livers of adult male mice (strain 129 SvJ), adult male rats (Sprague Dawley), and adult male golden hamster as described previously (18). DNA (15 ␮g) was digested with 40 U HindIII (Promega Corp.), electrophoresed on a 0.8% (wt/vol) agarose gel, and then incubated in 0.25 n HCl for 10 min, in 0.5 n NaOH and 1.5 m NaCl for 30 min, and twice in 0.5 m Tris-HCl (pH 7.5), 1 mm EDTA, and 1.5 m NaCl for 15 min each time. DNA fragments were transferred overnight to a Hybond N⫹ nylon membrane (Amersham Pharmacia Biotech) by blotting (19). The membrane was baked 2 h at 80 C and prehybridized for 3 h at 42 C in 6 ⫻ SSC (standard saline citrate), 1% (wt/vol) SDS, 100 ␮g/ml salmon sperm DNA, 50% (vol/vol) formamide, and 5% (wt/vol) dextran sulfate. The random primed 32P-radiolabeled probe was synthesized using the Rediprime DNA labeling system (Amersham Pharmacia Biotech) and incubated overnight (106 dpm/ml). The filter was washed once in 2 ⫻ SSC for 15 min, once in 2 ⫻ SSC and 0.1% (wt/vol) SDS for 15 min, once in 2 ⫻ SSC and 0.1% (wt/vol) SDS for 30 min, once in 0.2 ⫻ SSC and 0.1% (wt/vol) SDS for 15 min at 65 C, and once in 0.1 ⫻ SSC and 0.1% (wt/vol) SDS for 15 min at 65 C before being autoradiographed for 0.5– 4 days at ⫺80 C with Hyperfilm MP film (Amersham Pharmacia Biotech).

Northern blotting Total RNA (10 ␮g) was denatured for 15 min at 65 C and cooled on ice. RNA samples were loaded on a 1% (wt/vol) agarose gel containing 20 mm MOPS (pH 7), 5 mm sodium acetate, 1 mm EDTA, and 6% (vol/vol) formaldehyde and then transferred to a Hybond N⫹ nylon membrane (Amersham Pharmacia Biotech) by blotting overnight in 20 ⫻ SSC. The membrane was washed once in 2 ⫻ SSC, dried, and baked 2 h at 80 C. The prehybridization was carried out for 3 h at 42 C in 50% (vol/vol) formamide, 6 ⫻ SSC, 5 ⫻ Denhardt’s solution, 100 ␮g/ml salmon sperm DNA, and 0.1% (wt/vol) SDS and then random primed 32P-labeled mE-RABP cDNA prepared with the Random Prime DNA labeling kit (Amersham Pharmacia Biotech) was added and incubated overnight. The filter was washed once in 2 ⫻ SSC for 15 min, once in 2 ⫻ SSC and 0.1% (wt/vol) SDS for 15 min, once in 2 ⫻ SSC and 0.1% (w/vol) SDS for 30 min, once in 0.2 ⫻ SSC and 0.1% (wt/vol) SDS for 15 min, and once in 0.l ⫻ SSC and 0.1% (wt/vol) SDS for 15 min at 65 C before being autoradiographed with Hyperfilm MP (Amersham Pharmacia Biotech). Northern blots were reprobed with a cloned 18S cDNA to standardize the loaded RNA samples. The relative absorbance of the mEP17 and 18S RNA was determined using an imaging densitometer (model GS-670, Bio-Rad Laboratories, Inc., Richmond, CA) and the application Molecular Analyst.

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In situ hybridization Nonisotopic in situ hybridization (20, 21) was performed on 4- to 6-␮m thick cryosections of fresh-frozen mouse epididymides. Sections were fixed in 4% formaldehyde in 0.1 m sodium phosphate buffer, pH 7.2, and then incubated for 10 min in PBS containing 5 ␮g/ml proteinase K. After two rinses in PBS, sections were incubated in 0.25% acetic anhydride in 0.1 m triethanolamine, pH 8.0, for 15 min. Sense and antisense riboprobes were

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prepared in 20-␮l transcription reactions containing SP6 (Promega Corp.) or T7 (New England Biolabs, Inc., Beverly, MA) polymerase; 1 ⫻ transcription buffer; 1 mm each of ATP, CTP, and GTP; 0.65 mm UTP; 0.35 mm digoxigenin-UTP (Roche, Indianapolis, IN); and 1 ␮g linearized F3 plasmid carrying the mEP17 cDNA. Unincorporated nucleotides were removed on a Chroma Spin-100 500 mm NaCl, 20 mm Tris-HCl (pH 7.5), and 1 mm EDTA (STE) column (CLONTECH Laboratories, Inc., Palo Alto, CA).

FIG. 1. Genomic organization of the mEP17 gene. The mEP17 gene is located upstream from the mE-RABP gene within the locus [A3, B] of mouse chromosome 2. Exon sizes are indicated in nucleotides. The distance between each exon and the major transcription initiation site (⫹1 nt) of the mE-RABP gene is indicated in kilobases. The major transcription initiation sites of both genes are represented with broken arrows. Primers FwmEP17cDNA and RiIII, used to amplify the full-length mEP17 cDNA by 3⬘-RACE, are described. Two important motifs, G-X-W and T-D-Y, and two cysteine residues (C) that are believed to be important for the three-dimensional structure of lipocalin proteins are also indicated.

FIG. 2. Nucleic acid sequence of the full-length cDNA encoding mEP17 (clone F3). The putative signal sequence is boxed and shaded. The conserved motifs G-X-W and T-D-Y and two cysteine residues conserved among members of the lipocalin superfamily are boxed. The putative polyadenylation signal 5⬘AATAAA-3⬘ is underlined. The 18-bp pyrimidine-rich region that is conserved between mEP17 and mE-RABP cDNA is indicated with dashed lines.

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NEW 17-kDa LIPOCALIN Digoxigenin riboprobes were denatured for 5 min at 80 C; diluted in hybridization buffer composed of 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, 4 ⫻ SSC, 1 ⫻ Denhardt’s reagent, and 0.5 mg/ml yeast transfer RNA; and incubated with the sections overnight at 55 C. The slides were washed at room temperature for 5 min in 2 ⫻ SSC, rinsed in STE, and then incubated for 30 min in STE containing 40 ␮g/ml ribonuclease A. The

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sections were washed sequentially for 5 min each in 2 ⫻ SSC and 50% formamide at 50 C, then at room temperature with 1 ⫻ SSC, and finally with 0.5 ⫻ SSC. To detect hybridized probes slides were rinsed in 100 mm Tris-HCl (pH 7.5) and 150 mm NaCl, blocked for 1 h in 100 mm Tris-HCl (pH 7.5) and 150 mm NaCl containing 2% horse serum and 0.1% Triton X-100 (blocking solution), and incubated for 1 h in 1:500 diluted alkaline phosphatase-conjugated antidigoxigenin (Roche) in blocking solution. Slides were rinsed three times in blocking solution and then in a substrate buffer of 100 mm Tris-HCl (pH 9.5), 100 mm NaCl, and 50 mm MgCl2. Color development was performed in substrate buffer containing 0.17 mm 5-bromo-4-chloro-3-indolyl phosphate, 10 mm N-ethyl-maleimide, and 1 mm levamisole as an inhibitor of endogenous alkaline phosphatase. Color development was stopped with 10 mm Tris-HCl (pH 8.0) and 1 mm EDTA. Sections were examined and photographed with a Carl Zeiss Axiophot (New York, NY) using both brightfield and phase contrast optics.

Results Identification of the mEP17 gene

FIG. 3. Hydropathic analysis (Kyte and Doolittle) of mEP17. The 21 first amino acid residues of mEP17 encode a highly hydrophobic domain of 2.4 kDa (black area). The strict cut-off limit (DAS; transmembrane prediction server) for signal peptide is indicated by dashed lines. The positions of amino acids are indicated at the bottom of the graph.

We have recently shown that 5 kb of the 5⬘-flanking region of the mE-RABP gene targets a high level of expression of a foreign gene to the principal cells of the mid/distal caput epididymidis in transgenic mice (16). To identify cis-DNA regulatory elements required for tissue- and region-specific expression of the mE-RABP gene, this 5-kb promoter fragment was entirely sequenced. A computer-based analysis of

FIG. 4. The amino acid sequence of mEP17 was compared with the GenBank database and was found similar to members of the lipocalin superfamily [hoLAC, horse ␤-lactoglobulin (25); QSP, quiescence-specific protein (26); mE-RABP, murine epididymal retinoic acid-binding protein (12); rat ERABP, rat epididymal retinoic acid-binding protein (5); PP14, human placenta protein of 14 kDa (27); MUP4, mouse urinary protein 4 (28); LESPIV, lizard epididymal secretory protein IV (29)].

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the nucleotide sequence revealed the presence of an open reading frame (ORF) encoding 34 amino acid residues. Analysis of this ORF revealed weak homology with the mE-RABP protein itself in the domain encoded by exon 4. This exon is well conserved in size among genes encoding lipocalins and encodes the motif T-D-Y, which is highly conserved among the lipocalins and is believed to be involved in the threedimensional structure of these proteins. We hypothesized that this ORF was part of a gene encoding a new lipocalin and that the genomic organization of this gene would be conserved as in other members of the lipocalin superfamily. Thus, we predicted the localization of several exons, including exon 1 (Fig. 1). The DNA sequence of the predicted exons allowed us to design a DNA primer (FwmEP17cDNA), which was used to clone the corresponding cDNA by rapid amplification of 3⬘-cDNA ends (3⬘-RACE; Fig. 1). As the 5-kb genomic DNA sequence restricted gene expression to the epididymis, epididymal total RNA was reverse transcribed using the RACE III primer. A full-length cDNA (694 bp) was generated by PCR using the FwmEP17cDNA and RiIII primers (Fig. 2). The exon/intron boundaries were confirmed by comparison of sequences between the cDNA and the genomic DNA. The genomic organization, constituted by seven exons interrupted by six introns, was identical to that of the mE-RABP gene. Moreover, our results indicated that the exon/intron boundaries were also strictly conserved between the genes. We also noticed that a short 18-bp pyrimidine-rich nucleotide sequence within the 3⬘-untranslated region was conserved between mEP17 and mE-RABP cDNA (Fig. 2). Analysis of the ORF

The ORF deduced from the 694-bp cDNA encodes 175 amino acids of an estimated 19.8-kDa polypeptide at pI 5.69 (Fig. 2). The analysis of the hydropathic pattern of the precursor indicated that the first 21 amino acid residues constituted a transmembrane domain (Fig. 3). This 2.4-kDa domain is probably a signal peptide, because it is 1) highly hydrophobic, 2) similar to the signal peptide of the mE-RABP protein, and 3) in agreement with the sliding window/matrix scoring method and ⫺1, ⫺3 rule for predicting signal peptide cleavage (22). This observation implies that the new gene encodes a putative 17-kDa mature protein (17.39582– 17.61202 kDa; pI 5.69 –5.94) that may be secreted into the luminal fluid. Therefore, we named the new gene, mEP17, for mouse epididymal protein of 17 kDa. The mEP17 amino acid sequence was compared with the GenBank database (Fig. 4). It is well documented that members of the lipocalin superfamily share a few highly conserved amino acid sequence motifs believed to be important for the tertiary structure of the proteins, but have low overall sequence identity (⬃20%) and similarity (⬃50%) (23, 24). As expected, low, but significant, identity was found with members of the lipocalin superfamily, including horse ␤lactoglobulin (24.9% identity) (25), chicken quiescencespecific protein (24.3%) (26), mE-RABP (23.9%) (12), human placenta protein of 14 kDa (23.4%) (27), ESP I (22.9%) (5), murine urinary protein IV (21%) (28), and lizard epididymal secretory protein IV (15.5%) (29). The mEP17 protein pos-

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sesses the three structurally conserved regions (T-D-Y, GX-W and two cysteine residues) that have been proposed to be a prerequisite for a protein to be considered a lipocalin (30). These motifs are encoded by exons 4, 2, 3, and 6, respectively, as reported previously for other genes encoding lipocalins. Analysis of the promoter region

The transcription initiation sites of the mEP17 gene were determined by primer extension using epididymal total RNA as template and the mEP17PE2 primer located in the first exon (Fig. 5). Two major transcription initiation sites were localized 22 and 18 nucleotides from the putative translation initiation site and were numbered ⫹1 and ⫹5, respectively. Two minor transcription initiation sites were also detected at positions ⫹2 and ⫹4. A putative TATA box (5⬘-TATAAG-3⬘) and a CAAT box (5⬘-CCAAT-3⬘) are present at positions ⫺26 and ⫺73, respectively (Fig. 6). A putative SP 1 transcription factor-binding site is located at position ⫺240. A putative retinoic acid response element (RARE) constituted by a direct repeat separated by 1 bp (DR1) is present at position ⫺259. A 2.5-kb EcoRI restriction fragment overlapping with the pHindIII clone was isolated from the genomic BAC clone 10983 (15) to characterize 1.9 kb of the 5⬘-flanking region of the mEP17 gene (Fig. 6). A computer-base analysis of the DNA sequence revealed the presence of a putative androgen receptor-binding site at position ⫺342. The presence of several putative activator protein-1 and c-Ets cis-DNA regulatory elements as well as several potential sex-determining region Y gene product-related transcription factor-binding sites was also noted.

FIG. 5. Primer extension analysis of the 5⬘-end of the mEP17 mRNA. Total RNA extracted from the epididymis or transfer RNA was reverse transcribed with the 32P-radiolabeled mEP17PE2 primer and extended using AMV RT as described in Materials and Methods. Lanes CTA and G are the 35S-radiolabeled DNA-sequencing reactions carried out using the mEP17PE2 primer and the pHindIII clone as template. The localizations of two major (arrows) and two minor (arrowheads) transcription initiation sites are indicated.

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FIG. 6. Localization of putative cis-DNA regulatory elements within the 5⬘-flanking region of the mEP17 gene. Broken arrows and arrowheads indicate the major and minor transcription initiation sites, respectively. The computer analysis was carried out using TFSEARCH version 1.3 (Yutaka Akiyama: TFSEARCH: Searching Transcription Factor Binding Sites, http://www.rwcp.or.jp/papia/). ARBS, Androgen receptor-binding site; RARE, retinoic acid response region; SP1, stimulating protein 1; SRY, sex-determining region Y gene product; C/EBP, CCAAT/enhancerbinding protein; AP-1, activator protein 1; AP-4, activator protein 4; Sox-5, SRY-related HMG box gene 5.

Another gene related to the mEP17 gene or a pseudogene may be present in the mouse genome

The 694-bp 32P-radiolabeled mEP17 cDNA was used to probe the mouse strain 129 genomic DNA in Southern blot experiments (Fig. 7). A complex hybridization pattern was obtained with most of the restriction enzymes used. In particular, two bands (3.8 and 9 kb in size) were detected when the HindIII restriction enzyme was used. The 9-kb HindIII restriction fragment, but not the 3.8-kb DNA fragment, was in agreement with the restriction map of the mEP17 gene locus. The 3.8-kb HindIII restriction fragment could not be explained by the presence of a different allele of the mEP17 gene, because this DNA fragment was not detected when the

mE-RABP cDNA or the mE-RABP promoter was used as probe (15). In addition, hybridization of the plasmid Bac 10983, containing 175 kb of the mEP17 gene locus, with the mEP17 cDNA used as probe revealed the presence of a 9-kb HindIII restriction fragment only (not shown). Therefore, it is likely that the HindIII 3.8-kb restriction fragment belongs to a pseudogene or to another gene encoding a protein related to mEP17. Tissue-, region-, and cell-specific expression of the mEP17 gene

The tissue distribution of mRNA encoding the mEP17 protein was examined by Northern blot analysis of total

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RNA from 12 different tissues, including the spleen, liver, heart, lung, brain, kidney, testis, epididymis, vas deferens, seminal vesicles, uterus, and ovary (Fig. 8A). Hybridization of the Northern blots with a 32P-radiolabeled mEP17 cDNA probe revealed two RNA species of about 3.1 and 1 kb only in the epididymis (Fig. 8). The total length of the mEP17 gene, including exons and introns, is 3.1 kb. To determine whether the 3.1-kb RNA could be the precursor RNA, two epididymal RNA samples were run side by side and hybridized individually with the cDNA probe or with a probe encompassing intron 1 of the mEP17 gene (Fig. 8B).

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The intron 1 probe hybridized with the 3.1-kb RNA, but not the 1-kb RNA, indicating that the 3.1-kb RNA may be an unspliced precursor. In situ hybridization of mEP17 mRNA was carried out using sense and antisense digoxigenin-labeled riboprobes generated from the mEP17 cDNA. The mEP17 mRNA was detected only in the principal cells of the initial segment of the epididymis and is localized basally (Figs. 9 and 10). No hybridization was seen when the sense riboprobe was used (Fig. 10). mEP17 gene expression was high throughout the initial segment and decreased progressively at the beginning of segment 2. A checkerboard pattern was observed in segment 2, i.e. some epithelial cells expressed high levels of mEP17 transcripts, and other did not. No mEP17 mRNA was detected in the efferent ducts, mid and distal caput, or corpus and cauda epididymidis using sense or antisense riboprobes. Together our results demonstrate that the mEP17 gene is specifically expressed in the principal cells of the initial segment of the epididymis and that a large number of mEP17 transcripts are not spliced. Hormonal regulation of the mEP17 gene

FIG. 7. Southern blot analysis of mouse strain 129 genomic DNA. Genomic DNA was extracted from the liver and digested with appropriate restriction enzymes (top). DNA fragments were separated on an agarose gel, transferred to a nylon membrane, and hybridized using the 32P-radiolabeled mEP17 cDNA as probe. A ladder of sizes (kilobases) is presented on the left.

The effect of castration on mEP17 mRNA expression was studied by Northern blot analysis (Fig. 11A). Total RNA extracted from the epididymis at 5, 10, 20, and 30 days postcastration was hybridized with the 32P-radiolabeled mEP17 cDNA probe. High levels of mEP17 transcripts were detected in intact animals as described above. Castration led to a rapid decrease in mEP17 gene expression, as no mEP17 transcript could be detected at 5 days postcastration. To study the hormonal regulation of mEP17 mRNA expression, unilateral castration for 4 days was carried out (Fig. 11B). Northern blot analyses showed that mEP17 mRNA expression levels strongly decreased in the epi-

FIG. 8. A, epididymis-specific expression of the mEP17 gene. Total RNA (10 ␮g/lane) was extracted from different male and female tissues and hybridized with the 32P-labeled mEP17 cDNA. Two major transcripts of 1 and 3.1 kb were detected only in the epididymis. B, Hybridization of total RNA (10 ␮g/lane) extracted from the epididymis, with the 32 P-labeled intron 1 of the mEP17 gene or with the 32P-labeled mEP17 cDNA used as probes. Only the 3.1-kb transcripts were detected with the intron 1 probe, suggesting that these transcripts are probably unspliced mEP17 precursor RNA.

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well. Our result suggests that an mEP17-like gene has been conserved among the rodents and therefore suggests that mEP17 may play an important function in male fertility. Discussion mEP17 is a new member of the lipocalin superfamily

In this study we identified within the 5⬘-flanking region of the mE-RABP gene, a gene encoding a precursor protein of 19.8 kDa. The analysis of the amino acid sequence of the precursor, as deduced from the open reading frame of the cDNA, showed that it contains two motifs (G-X-W and TD-Y) and two cysteine residues that are particularly well conserved among members of the lipocalin superfamily (23). With the exception of these motifs, mEP17 showed low sequence similarities with the other known lipocalins. However, it is well established that membership in the lipocalin superfamily is based not on sequence homology but, rather, on secondary and tertiary structure homology (only 25% identity and 50% homology, on the average, between members). The tryptophan residue of the motif G-X-W is required for the binding of lipophilic ligands. The two cysteine residues form the single intramolecular disulfide bond of the lipocalins. This disulfide bridge is required to maintain the conformation of the native lipocalin in the presence of the ligand and influences the ligand affinity. In addition, the presence of a putative signal sequence of 2.4 kDa at the N-terminal end of the precursor suggests that it is cleaved to generate a 17-kDa mature secretory protein. This observation is consistent with the fact that lipocalins are extracellular proteins found in most body fluids (30). FIG. 9. Region-specific expression of the mEP17 gene. In situ hybridization of mEP17 transcripts was carried out using a digoxigeninlabeled antisense mEP17 RNA. The mEP17 mRNA was highly expressed only in the principal cells of the initial segment (IS) of the caput epididymidis. Then a checkerboard pattern (some cells expressed high levels of mEP17 transcripts, whereas other did not; see arrow) was observed at the boundary between the initial segment and the proximal caput epididymidis. No staining was found in the efferent duct (ED) or mid and distal caput epididymidis (Cp).

didymis from the castrated side, but not in that from the intact side. In addition, testosterone replacement for 10 days to 35-day-castrated mice did not restore mEP17 gene expression (Fig. 11C). Together our results demonstrate that mEP17 gene expression is not dependent on circulating androgens, but is dependent on a testicular factor(s) (that may include androgens) available from testicular fluid. mEP17 gene is conserved during evolution from rodents to hamster

To determine whether the mEP17 gene may be conserved during evolution, genomic DNA, extracted from mouse (129 SvJ), rat (Sprague Dawley), and hamster, was analyzed by Southern blots using the 32P-labeled mEP17 cDNA as a probe (Fig. 12). As described previously with the HindIII restriction digest, two DNA fragments (3.8 and 9 kb in size) were easily detected in the mouse genome. A 9-kb HindIII restriction fragment was also observed in the rat genome. A larger DNA fragment (⬎15 kb) was detected in the hamster genome as

Gene duplication gives rise to an epididymis-specific lipocalin multigene family

The genomic organization of the mEP17 gene, consisting of seven exons and six introns, is similar to that of genes encoding other lipocalins, including the complement component 8␥ (31), PGD2 synthase (32), and mE-RABP (15). Interestingly, these genes were also mapped to the proximal region of mouse chromosome 2 (15, 33). This suggests that they result from the duplication of a common ancestral gene within this chromosomal region. Furthermore, a phylogenetic comparison of the ORF and conservation of the exon/ intron boundaries between mEP17 and mE-RABP genes indicates that both genes may result from a more recent duplication. The conservation of a 18-bp pyrimidine-rich DNA region within the 3⬘-untranslated region of the mEP17 and mouse and rat E-RABP genes supports this assumption. However, one can also hypothesize that this DNA motif is conserved because it may be important for mRNA stability and/or translation. The 3⬘-untranslated region of mRNA contains an important cis-regulatory element(s) that binds specific stabilizing or destabilizing proteins. For instance, binding of poly(C)-binding protein to a pyrimidine-rich sequence, located within the 3⬘-untranslated region, has been associated with stability of mRNA encoding the human tyrosine hydroxylase or ␣-globin proteins (34, 35). Further studies will be required to determine whether the pyrimidine-rich sequence found in the 3⬘-untranslated region of the epididymis-specific lipocalins has a specific function.

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FIG. 10. Cell-specific expression of the mEP17 gene. Upper panel, High power magnification of the boxed region in Fig. 9 showing the boundary between the initial (IS) and proximal caput epididymidis (Cp). The mEP17 mRNA was highly expressed only in the principal cells of the initial segment. Note that mEP17 messengers were localized basally. No staining was detected in the conjunctive tissue (CT) or in the epithelial cells of the proximal caput epididymidis. Lower panel, Hybridization of a section of the initial segment with a sense digoxigenin-labeled mEP17 RNA. No signal was detected in the epithelial cells, demonstrating the specificity of the staining seen when the antisense mEP17 probe was used.

The presence of several lipocalins expressed specifically in the epididymis has previously been described in a lower vertebrate. The lizard epididymal secretory proteins (LESP)

family is composed of nine androgen-dependent polypeptides (LESP II–IX) that are immunologically related, with an apparent molecular mass of approximately 18 kDa (36).

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FIG. 11. Hormonal regulation of the mEP17 gene. A, Epididymal total RNA (10 ␮g/lane) was extracted from intact (I) and 5-, 10-, 20-, and 30 day-castrated (C5, C10, C20, and C30, respectively) animals and then hybridized with the 32P-labeled mEP17 cDNA (exposure time, 24 h). B, mEP17 mRNA expression levels were analyzed 4 days after hemicastration (exposure time, 48 h). Note that mEP17 mRNA level is much lower (0.7% of control) in the epididymis removed from the castrated side compared with that from the intact side. I, Intact animals; C5, 5-day-castrated animals; HI, epididymis extracted from the intact side of 4-day-hemicastrated animals; HC, epididymis extracted from the castrated side of 4-day-hemicastrated animals. C, mEP17 mRNA expression was analyzed in 5-day-castrated animals supplemented for 10 days with testosterone propionate (TP) or sesame oil (C5). Androgen replacement fails to restore mEP17 gene expression after castration (exposure time, 48 h).

These lizard proteins, synthesized in the epithelial cells of the caput epididymidis, are secreted into the luminal fluid. Some of these LESP proteins bind tightly to the head region of spermatozoa. N-Terminal microsequencing of the LESP proteins suggests that they are encoded by at least five distinct genes. Interestingly, the recent molecular characterization of LESP IV indicates that the L protein family belongs to the lipocalin superfamily (29). LESP IV is similar to mEP17 and, to a lesser extent, to mE-RABP. Taken together, these results suggest that mEP17 and mE-RABP genes are the first identified members of a multigene subfamily encoding epididymal lipocalins in the mouse. Although the PGD2 synthase (PGD2-S) gene is not specifically expressed in the caput epididymidis, it may be considered a member of this subfamily. The PGD2-S gene, also localized in the proximal segment of mouse chromosome 2, encodes another related lipocalin with multiple function. The PGD2-S protein catalyzes the conversion of the cyclooxygenase-derived intermediate PGH2 to PGD2 in the presence of sulfhydryl compounds. In addition to its enzymatic activity, PGD2-S binds retinal and retinoic acid, and therefore should be considered a putative retinoid transporter (37). More members of this multigene subfamily may remain to be identified. Expressions of mEP17 and mE-RABP genes are differently regulated

The mEP17 and mE-RABP genes are both expressed in the caput epididymidis and are placed in tandem on mouse chromosome 2. In situ hybridization experiments have shown that mEP17 gene expression was restricted to the principal cells of the initial segment of the caput epididymidis. Interestingly, this region-specific expression is complementary to that of the mE-RABP gene, as mE-RABP mRNA are detected only from the mid/distal caput epididymidis (12). Although mEP17 and mE-RABP genes may share

common cis-DNA regulatory sequences responsible for the tissue-specific gene expression, our observations suggest that distinct regulatory elements are responsible for the region-specific gene expression. Unlike mE-RABP, mEP17 gene expression almost disappeared 4 days after hemicastration from the castrated side, suggesting that gene expression was dependent on factors circulating within the testicular fluid. Previous studies have described histological modifications in the initial segment after efferent duct ligation (38). These changes are probably the consequence of altered gene expression. Indeed, numerous genes expressed in the initial segment and dependent on testicular factors other than androgens have been reported, including the ␥-glutamyl transpeptidase mRNA IV (39), polyomavirus enhancer activator 3 (40), A-raf (41), 5␣reductase (42), and CRES (43). Regulation of epididymal function by testicular factors remains poorly understood (44). Soluble components of rete testis fluid may regulate gene expression. For instance, a 43-kDa protein found within testicular fluid and immunologically related to basic fibroblast growth factor has been reported to regulate the GGT activity in the rat epididymis (45). In addition, expression of the proenkephalin mRNA may be regulated by spermassociated factors (46). Although a putative androgen response element is present within the mEP17 gene promoter, testosterone replacement to hemicastrated male mice failed to restore mEP17 gene expression. Testosterone may have no effect on mEP17 gene transcription, but one cannot exclude that the androgen receptor requires cooperation with other transcription factors induced by an extracellular signal present within the luminal fluid. A putative RARE is present at position ⫺259 nt within the mEP17 gene promoter. Most genes encoding a retinoid-binding protein show the presence of a functional RARE in the promoter region. For instance, RAREs have been identified

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0.2, 0.6, 1, and 5.6 kb upstream the promoter of the human retinol-binding protein (47), human cellular retinol binding protein type II (CRBP-II) (48), rat CRBP-I (49), and human CRABP-II (50) genes, respectively. Thus, mEP17 gene expression may be regulated by retinoic acid. Putative function of the mEP17 protein

FIG. 12. Southern blot analysis of mouse (strain 129), rat (Sprague Dawley), and hamster (golden) genomic DNA (15 ␮g/lane) digested with HindIII. DNA fragments were separated on an agarose gel, transferred to a nylon membrane, and hybridized using the 32Pradiolabeled mEP17 cDNA as probe. A ladder of sizes (kilobases) is presented on the left. A mEP17-like gene was detected in rat and hamster genomes.

Because we still do not know what ligands bind to mEP17, all discussion of function, although based upon the amino acid sequence homology and the region-specific expression pattern, remains hypothetical. Lipocalins are primarily extracellular transport protein found in most body fluids and involved in the delivery of small lipophilic ligand (30). As mEP17 presents high homology with other lipocalins that bind to retinoids, the most plausible function for mEP17 is as a transport protein for retinoids within the epididymis. There is evidence that retinoids play an important role in regulating gene expression in the epididymis (51). Moreover, a vitamin A-free diet leads to pathological changes in the epididymal epithelium (52). The retinoids required for the epididymal function may have different sources. The retinol binding protein binds retinol that circulates in the plasma and is recognized by a membrane-bound receptor found on target cells (53, 54). Once retinol is internalized into the cells, it is bound to an intracellular binding protein termed CRBP-I. Within the mouse epididymis, CRBP-I is expressed in the initial segment (Zheng, W. L., unpublished data), suggesting that the uptake of retinol is likely in this segment. The CRBP-I expression pattern overlaps that of mEP17, suggesting that CRBP-I may deliver retinol or retinol-derived metabolites to mEP17. Upon binding to mEP17, these retinoids may be exported out of the cells and delivered to downstream epididymal epithelial cells lining the duct or to spermatozoa. Indeed, retinoic acid and retinyl esters are present in rat epididymal spermatozoa (55). However, the former hypothesis appears more plausible, as the overexpression of a dominant negative retinoic acid receptor ␣ in transgenic mice results in a loss of the cauda epithelium integrity (3). This demonstrates that this segment is highly dependent on retinoic acid. In addition, mEP17 may capture retinoids from the spermatozoa and release it to the epididymal epithelial cells lining the duct. This would imply that sperm-derived retinoids may play the role of a sperm-associated signal that regulates epididymal gene expression. Such a model has been proposed for regulation of proenkephalin gene expression in the rat caput epididymidis (46). Finally, other functions have been assigned to lipocalins, including maintenance of blood-organ barriers, regulation of the immune response, and mediation of cellular homeostasis (30). In conclusion, mEP17 may play an important role in the sperm maturation process or in the paracrine regulation of the epididymal function. Such roles have also been proposed for mE-RABP (14, 56). However, it is unlikely that mEP17 and mE-RABP functions are redundant, because of the different region-specific expression patterns. This, then, implies that epididymal lipocalins bind to different ligands or to the same

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ligand, but localize/transfer that ligand to different cellular compartments. Acknowledgments We thank the Cancer Center DNA Sequencing Core directed by Dr. K. Bhat for valuable technical assistance.

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