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Endocrinology Vol. 144, No. 3 877-886
Copyright © 2003 by The Endocrine Society

ARTICLE

The 5'-Flanking Region of the Murine Epididymal Protein of 17 Kilodaltons Gene Targets Transgene Expression in the Epididymis

Kichiya Suzuki, Yoshihiko Araki1, Mei-Ying Zhu, Jean-Jacques Lareyre, Robert J. Matusik and Marie-Claire Orgebin-Crist

Center for Reproductive Biology Research (K.S., Y.A., M.-Y.Z., R.J.M., M.-C.O.-C.), Departments of Obstetrics and Gynecology (K.S., Y.A., M.-Y.Z., M.-C.O.-C.), Urologic Surgery (R.J.M.), Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-2633; and Institut National de la Recherche Agronomique (J.-J.L.), 35042 Rennes Cedex, France

Address all correspondence and requests for reprints to: Kichiya Suzuki, Center for Reproductive Biology Research, Vanderbilt University, School of Medicine, Medical Center North C-3306, Nashville, Tennessee 37232-2633. E-mail: kichiya.suzuki{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A murine epididymal retinoic-acid-binding protein (mE-RABP) is specifically expressed in the mid/distal caput epididymidis and is androgen regulated. The murine epididymal protein of 17 kDa (mEP17) gene, a novel gene homologous to mE-RABP, is located within 5 kb of the 5'-flanking region of the mE-RABP gene. In contrast, expression of the mEP17 gene is restricted to the initial segment and regulated by factor(s) contained in testicular fluid. To identify cis-DNA regulatory element(s) involved in the tissue- and region-specific expression of the mEP17 gene in transgenic mice, we have studied the expression of a transgene containing 5.3 kb of the 5'-flanking region of the mEP17 gene (5.3mEP17) linked to chloramphenicol acetyltransferase (CAT) reporter gene. Significant caput epididymidis-specific CAT activity was detected in transgenic mouse lines; and CAT gene expression is restricted to the initial segment, as is the expression of the endogenous mEP17 gene. Ontogenic expression and testicular factor dependency also mimic that of endogenous mEP17 gene. These results suggest that the 5.3mEP17 fragment contains all the information required for spatial and temporal expression in the mouse epididymis. The 5.3mEP17 fragment will be useful to express a foreign gene of interest in the epididymis in an initial segment-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EPIDIDYMIS is essential for sperm maturation during sperm transit from testis to vas deferens (1, 2). In general, the epididymis is subdivided into four major segments designated as initial segment, caput, corpus, and cauda. The principal cells in each region have different functions (3). Numerous epididymal proteins are regionally expressed and secreted (4). Several genes, including proenkephalin (5), cystatin-related epididymal specific protein (6), 5{alpha}-reductase (7, 8, 9), and {gamma}-glutamyl transpeptidase (GGT) mRNA IV (10), are expressed in the initial segment and regulated by still-unidentified testicular factor(s) (11).

Previous studies from our laboratory have demonstrated that the murine epididymal retinoic-acid-binding protein (mE-RABP), a protein which is synthesized by the principal cells of the mid/distal caput epididymidis, is secreted into the lumen (12, 13). This molecule belongs to the lipocalin protein superfamily, which binds small hydrophobic molecules such as retinoids (14, 15, 16). The mE-RABP gene is located on mouse chromosome 2 [A3-B] (17). Recent data have shown that chromosome 2 [A3-B] contains a lipocalin cluster (18) that encompasses prostaglandin D2 synthase (19), complement component 8 {gamma} (20), mE-RABP (17), and 24p3 (21). Although prostaglandin D2 synthase is expressed in brain and epididymis and 24p3 is expressed in uterus, vagina, and epididymis, the mE-RABP gene is expressed only in the epididymis. To investigate the tissue-, region-, and cellspecific expression of the mE-RABP gene, we have established transgenic mice carrying 5 kb of the 5'-flanking region of the mE-RABP gene ligated to chloramphenicol acetyltransferase (CAT) reporter gene (22). In the transgenic mice, the reporter gene expression was restricted to the principal cells of the mid/distal caput epididymidis (22).

Recently, we found another gene, named the murine epididymal protein of 17 kDa (mEP17), encoding a 17-kDa protein, with its last exon only 1.7 kb upstream from the transcription initiation site of the mE-RABP gene. This 17-kDa protein also belongs to the lipocalin superfamily. The mEP17 gene is specifically expressed in the initial segment and is regulated by testicular factor(s) (23).

To identify cis-DNA regulatory element(s) involved in the tissue-specific expression of the mEP17 gene, we have studied the expression of a transgene containing 5.3 kb of the 5'-flanking region of the mEP17 gene (5.3mEP17) linked to the CAT reporter gene. In this study, we demonstrate that the 5.3mEP17 fragment can drive the specific expression of the CAT reporter gene to the initial segment, thereby mimicking the expression of the endogenous gene. We also demonstrate that the 5.3mEP17 fragment contains all of the information required for the spatial and temporal gene expression and the testicular factor(s)-dependent regulation of the mEP17 gene in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
B6D2F1 mice were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). All mice were housed under a constant 12-h light, 12-h dark cycle and were allowed free access to food and water. Orchiectomies were carried out by the scrotum route after inhalation of IsoVet (Isoflurane; Schering Plough Animal Health Corp., Union, NJ) or Ketamine/Xyladine ip injection (1 mg each per gram body weight; Fort Dodge Animal Health, Fort Dodge, IA). All experiments were conducted in accordance with the National Institutes of Health Guidelines for Care and Use of Animals in the Laboratory.

Chimeric construct
A DNA fragment containing the mEP17 5'-flanking region was isolated from BAC clone 10983 (17). In brief, 10 µg BAC clone DNA was digested with 30 U EcoRV, then the DNA fragments were ligated to pZErO-2.1 (Invitrogen, San Diego, CA). After transformation into competent cells, positive clones were screened by colony hybridization using 32P-labeled HindIII-EcoRI fragment containing mEP17 exon 1 (probe 1, Fig. 1AGo). A positive clone containing a 6.3-kb genomic fragment, named E57, was used to construct a chimeric plasmid. A 5.3-kb DNA fragment encompassing the 5'-flanking region of the mEP17 gene was generated from the E57, using appropriate restriction enzymes. To generate SalI site at position +22, PCR was performed using a primer pair: 5'-GGTATAAGGTTCCTGGTTGG-3', corresponding to -369 to -350 of the mEP17 gene sense sequence; and 5'-GGGTCGACCTCAGGGCCTGGCTTG-3', corresponding to +7 to +22 antisense sequence plus the SalI digestive site at the 5' end of the primer (underlined). Next, pBS57 was produced as follows. E57 was digested with EcoRV, to cut out the 6.3-kb fragment of the mEP17 gene, and the digested fragment was subcloned into pBluescript II SK(+) (Stratagene, La Jolla, CA). The PCR product (399-bp) was subcloned into pGEM-T Easy (Promega Corp., Madison, WI) and digested with SalI and HindIII, and then the digested 332-bp-length fragment was subcloned into SalI and HindIII digested pBS57. Finally, a 1680-bp CAT gene fragment was cut out from pBLCAT3 with SalI and KpnI, and the fragment was subcloned into SalI and KpnI sites of pBS57 to obtain pCAT5.3EvS (Fig. 1AGo).



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  		Figure 1. Schematic map of the chimeric construct and characterization of 5.3mEP17-CAT transgenic mouse lines. A, Restriction map of the mEP17 and mE-RABP gene locus and chimeric construct. Exons of the mEP17 and the mE-RABP genes are indicated by boxes. Subclones E57 and pHindIII cover 5.3 kb 5'-flanking region and all the exons of the mEP17 gene. The schematic representation of the chimeric construct, pCAT5.3EvS, was used for transgenic analysis of the mEP17 transcription regulatory region. The 5.3-kb fragment of the mEP 17 gene was ligated in front of the CAT gene to generate the 5.3mEP17-CAT transgene. EcoRV and KpnI were used to release the DNA fragment for pronuclei injection experiments. B, Detection of the transgene by PCR using tail DNA. Primers 1 and 2 were used to amplify a 329-bp DNA fragment corresponding to the transgene. The casein primers were used as a positive control of the PCR. WT, Wild-type mouse (B6D2F1). C, Southern blot analysis of genomic DNA extracted from wild-type and transgenic mouse lines. Twenty micrograms of genomic DNA were digested with HindIII, and resulting DNA fragments were separated on a 0.8% agarose gel. DNA fragments were blotted on a nylon membrane and hybridized with the 32P-labeled probe 2. Hybridization signals were exposed to x-ray film for 22 h.

 
DNA sequence analysis
All nucleotide sequences were determined on both strands, using the dideoxynucleotide chain termination method according to the manufacturer’s protocol (PE Applied Biosystems, Foster City, CA). The fluorescent-labeled reaction products were analyzed using DNA Sequencer (Model 377 or 3700, PE Applied Biosystems). All collected sequence data were analyzed and aligned using Vector NTI (InfoMax, North Bethesda, MD).

Transgenic mice
The 5.3-kb DNA fragment of the mEP17 gene driving the CAT reporter gene was excised from the pCAT5.3EvS using EcoRV and KpnI. The DNA fragment was purified on a 0.8% (wt/vol) low-melting-point agarose gel using the AgarACE enzyme (Promega Corp.). Transgenic mice (B6D2F1) were generated by microinjection of the DNA into the male pronucleus of a fertilized oocyte, using standard techniques (24). Transgenic animals were identified by PCR-based screening assay using isolated tail genomic DNA. Approximately 1 cm of the tail was digested overnight at 55 C in a proteinase K digestion mix containing 10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 25 mM EDTA, 1% sodium dodecyl sulfate, and 0.5 mg/ml proteinase K. Then DNA was extracted with 1 vol phenol/chloroform/isoamyl alcohol (25/24/1) and precipitated at room temperature with 2 vol absolute ethanol. Samples were centrifuged at 10,000 x g at 4 C for 15 min, washed with 70% ethanol, centrifuged at 10,000 x g at 4 C for 15 min, and dried for 2 h at room temperature. One hundred nanograms of genomic DNA were mixed with 1x PCR buffer, 2 U Taq DNA polymerase (Promega Corp.), 1.5 mM MgCl2, 0.2 µM concentration of each primer (MEP17TR, 5'-TGGTTGGTGCAGTCTTACTGC-3'; CAT-Rev, 5'-CAACGGTGGTATATCCAG-TG-3'; Casein-Fw, 5'-GATGTGCTCCAGGCTAAAGTT-3'; and Casein-Rv, AGAAACGGAATGTTGTGGAGT-3') and 0.2 mM deoxynucleotide triphosphate. DNA fragments were amplified for 30 cycles (95 C, 1 min; 50 C, 45 sec; 72 C, 45 sec) and one cycle (95 C, 1 min; 50 C, 45 sec; 72 C, 10 min) using thermal cycler (GeneAmp PCR system 9700; Perkin-Elmer Corp., Boston, MA). PCR products were analyzed on a 1% (wt/vol) agarose gel. To monitor CAT activity, organs were homogenized with a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) in 1–3 ml of 0.1-M Tris-HCl (pH 7.8), 0.1% Triton X-100. Insoluble material was removed by centrifugation (14,500 x g, 15 min, 4 C), and CAT assays were performed as described previously (25).

Southern blot analysis of genomic DNA
Genomic DNA was extracted from livers of wild-type or transgenic mouse lines as described previously. Twenty micrograms of genomic DNA was digested with HindIII, and resulting DNA fragments were separated on a 0.8% (wt/vol) agarose gel. DNA fragments were blotted on a Hybond N+ (Amersham Pharmacia Biotech, Piscataway, NJ) and hybridized with the 32P-labeled 321-bp probe 2 (Fig. 1AGo). Hybridization signals, observed after autoradiography, were quantified using a Densitograph version 4.0 (Atto Corp., Tokyo, Japan). The transgene copy number was determined by the following calculation: (ratio between the intensity of the DNA fragment corresponding to the transgenes and the 9-kb HindIII DNA fragment corresponding to the endogenous mEP17 gene) multiplied by two.

In situ hybridization
Tissues were fixed overnight in freshly prepared 4% (vol/vol) paraformaldehyde-PBS (pH 7.4), rinsed with PBS, and then dehydrated through a series of increasing concentrations of ethanol for a period of 3–5 h before embedding in paraplast. Tissues were sectioned at 7 µm. The CAT probe was prepared as described below. The CAT reporter gene was amplified from the pBLCAT3 vector using primers (5'-GGATCCATGGAGAAAAAAATCACTGG-3' and 5'-AGATCTTTAC-GCCCCGCCCTGCCA-3') and ligated into the pGEM-T easy vector to obtain the pGEM-CAT construct. CAT RNA probes were transcribed from pGEM-CAT and labeled with [35S]uridine 5'-triphosphate to a specific activity of l.2 x l09 cpm/µg using the in vitro transcription kit MAXIscript (Ambion, Inc., Austin, TX). Hybridization was carried out at 50 C with 2 x 104 cpm/ml riboprobe overnight in 50% (vol/vol) formamide, 300 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10 mM NaH2PO4 (pH 6.8), 5 mM EDTA (pH 8.0), 0.2% (wt/vol) Ficoll 400, 0.2% (wt/vol) polyvinylpyrrolidone, 10% (wt/vol) dextran sulfate, 200 µg/ml yeast transfer RNA, and 50 mM dithiothreitol. Excess of riboprobe was removed by washing in 2x saline sodium citrate (SSC), 20 mM ß-mercaptoethanol (ß-ME) for 15 min at 50 C once; followed by two washes in 4x SSC, 50% (vol/vol) formamide, 20 mM ß-ME for 30 min each at 55 C; and two washes in 4x SSC, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA for 10 min each at 37 C. Single-stranded RNA was digested in 4x SSC, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, and 20 µg/ml ribonuclease A for 30 min at 37 C. The reaction was stopped by two washes in 4x SSC, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA for 10 min each at 37 C and two washes in 4x SSC, 50% (vol/vol) formamide, 20 mM ß-ME for 30 min each at 55 C. Slides were quickly rinsed twice in H2O and air-dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed for 1–7 d at 4 C, developed, fixed, and mounted with Permount (Fisher Scientific, Pittsburgh, PA) for photography.

Northern blot analysis
Total RNA samples from 3–10 epididymides were extracted using RNeasy Midi Kit. Total RNAs were separated on 1% agarose gel and blotted to Hybond N+ by a capillary method (26). Twenty-five nanograms of the mEP17 cDNA (23) were labeled with [32P] deoxy-CTP using rediprime II (Amersham Pharmacia Biotech). Hybridization and serial washing was performed as previously described (27). The signals were visualized by exposure to Bio-Max MR (Eastman Kodak Co.).

Statistical analysis
The Mann-Whitney U test was used to examine the statistical significance of difference in CAT activity of the transgenic mice. The data were expressed as means ± SE from at least three individuals. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of transgenic mice carrying the CAT reporter gene driven by the 5.3mEP17 fragment
To assess whether the 5.3mEP17 fragment could direct the expression of a CAT reporter gene in the epididymis, transgenic mice were generated. To construct the transgene, the E57 clone containing a 6.3-kb genomic fragment encompassing the 5.3-kb 5'-flanking region and exon 1 of the mEP17 gene was isolated from BAC 10983. The 6.3-kb DNA fragment was sequenced on both strands. Finally, a 5.3-kb DNA fragment corresponding to the 5'-flanking region fragment of the mEP17 gene was isolated from the 6.3-kb genomic fragment and was ligated to the CAT gene to obtain the 5.3mEP17-CAT transgene (Fig. 1AGo). After pronuclei injection of the 5.3mEP17-CAT transgene and embryo transfer, 33 offspring were generated. Transgene integration was confirmed by PCR and Southern blotting analysis on genomic DNA. As can be seen in Fig. 1BGo, seven founder animals were PCR positive. The Southern blotting analysis showed that transgenes were randomly inserted into the mouse genome, and the transgene copy number integrated in the genome was determined by calculating the ratio between the hybridization signals of the transgene and the endogenous mEP17 gene (Fig. 1CGo). The 5.3mEP17-CAT transgene copy number in transgenic mice lines A, B, C, D, E, F, and G was 39, 6, 2, 1, 1, 17, and 18 copies, respectively.

Tissue distribution of CAT reporter gene activity
We have already reported that endogenous mEP17 mRNA is specifically expressed in the initial segment (23). To confirm that the 5.3mEP17 fragment can direct transgene expression in the initial segment, we first measured CAT activity of the lysates prepared from caput epididymidis of seven transgenic lines from 2- to 3-month-old mice. Among the seven transgenic mice lines, three lines (A, B, and F) demonstrated CAT activity in the caput epididymidis (Table 1Go). The level of reporter gene expression between transgenic mouse lines was not correlated to transgene copy number. However, in line F, increased CAT activity between heterozygous and homozygous was 2.8-fold, which is proportional to a theoretical increase of the copy numbers. Next, we examined tissue-specific expression of the 5.3mEP17-CAT transgene using the 3 lines that have high caput-specific CAT activity (line F, line A, and line B). No CAT activity was detected in the corpus and cauda epididymidis and in 15 other tissues, including testis, vas deferens, seminal vesicle, prostate, spleen, kidney, heart, lung, brain, stomach, small intestine, liver, muscle, bulbourethral gland, and bladder in line F and line A (Fig. 2Go). Similar tissue specificity was observed in line B (data not shown). Female tissues from line F, including ovary, oviduct, and uterus, had no CAT activity (Fig. 2Go). These results demonstrate that the 5.3mEP17 fragment has the ability to target the reporter gene expression specifically in the caput epididymidis.


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  		Table 1. TABLE I. CAT activity in the epididymis of heterozygous and homozygous 5.3mEP17-CAT transgenic mice

 


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  		Figure 2. Tissue-specific expression of the 5.3mEP17-CAT transgene in the caput epididymidis (epi.). Tissues were dissected from three mice (8–12 wk old) belonging to B6D2F1 wild-type (WT) or heterozygous transgenic mouse lines A and F. Indicated at the right are female tissues, including ovary, oviduct, and uterus from WT and line F. CAT activity was measured as described in Materials and Methods. Tissue lysates (200 µg protein) were used to determine the CAT activity in the caput epididymidis. Values are presented as the mean ± SE per milligram of protein. Bulboureth. gl., Bulbourethral gland; int., intestine.

 
Region-specific expression of the CAT reporter gene
We have previously reported that the endogenous mEP17 gene expression is restricted to the initial segment. To address the question of whether the 5.3mEP17 fragment contains all of the information required for region- and cell-specific expression, in situ hybridization of the CAT reporter gene was carried out. The tissue section of the epididymis obtained from line F was hybridized with CAT antisense probe. CAT mRNA was detected only in the principal cells of the initial segment (Fig. 3Go, A and C). Therefore, the spatial expression of the 5.3mEP17-CAT transgene is identical with that of the endogenous mEP17 gene (Fig. 3Go, B and D). No CAT mRNA was detected in the efferent ducts, the mid/distal caput, the corpus, and cauda epididymidis of 5.3mEP17-CAT transgenic mice. Although transgene expression was lower, the same initial segment-specific transgene expression was observed in line B (data not shown). Together, these results demonstrate that the 5.3mEP17 fragment contains all the cis-DNA regulatory elements required for tissue-, region- and cell-specific mEP17 gene expression.



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  		Figure 3. In situ hybridization of the 5.3mEP17-CAT transgene in the epididymis. A, Epididymis from heterozygous mouse line F was hybridized with 35S-labeled CAT antisense probe. Signal was detected only in the initial segment. B, Epididymis from wild-type (WT) mouse was hybridized with 35S-labeled mEP17 antisense probe. High magnification of the initial segment region from line F (C) and WT mouse (D). Note that the CAT gene expression is detected only in the principal cells of the initial segment in line F (C) and WT (D). Sections were exposed to emulsion for 1 d (C and D) or 7 d (A and B). A and B, Dark field; C and D, phase contrast; bars, 50 µm.

 
Testicular factor(s) regulation of the 5.3mEP17-CAT transgene
Endogenous mEP17 gene expression was shown to be regulated by testicular factor(s) (23). To determine whether the 5.3mEP17-CAT transgene is regulated by testicular factor(s), transgenic mice were orchiectomized, either bilaterally or unilaterally, under various conditions. The expression level of the transgene was monitored by CAT assay (Fig. 4AGo). One week after bilateral orchiectomy, CAT expression was decreased in transgenic mice. Testosterone replacement after bilateral orchiectomy did not restore transgene expression. Similarly, after unilateral orchiectomy, transgene expression was strongly decreased in the orchiectomized side, compared with the intact side. In situ hybridization was performed to confirm endogenous mEP17 expression in wild-type mice (Fig. 4Go, B and C) and CAT reporter gene expression in line F (Fig. 4Go, D and E). One week after unilateral orchiectomy, the intact side showed high-level expression of endogenous mEP17 in wild-type (Fig. 4BGo) and CAT transgene expression in line F (Fig. 4DGo); however, the orchiectomized side demonstrated a decreased endogenous mEP17 expression (Fig. 4CGo) and no CAT expression (Fig. 4EGo). Altogether, our data demonstrate that 5.3mEP17-CAT transgene expression is tightly regulated by testicular factors in a manner similar to that of the endogenous mEP17 gene.



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  		Figure 4. Testicular factor(s) regulation of the 5.3mEP17-CAT transgene in the epididymis of heterozygous mouse line F. A, 5.3mEP17-CAT expression after bilateral or unilateral orchiectomy. Three adult mice (8–10 wk old) were prepared for each treatment. CAT activity was determined in the epididymis of normal mice and heterozygous adult male mice line F, 1 wk and 2 wk (1W and 2W) after bilateral orchiectomy, 1 wk after bilateral orchiectomy and immediate supplementation with testosterone propionate (B+T) (2 µg/g body weight·d), 2 wk after bilateral orchiectomy with testosterone supplementation starting 1 wk after orchiectomy (T+), 1 wk after unilateral orchiectomy, orchiectomized side (Orchiec) and intact side (Intact), and compared with that of intact wild-type adult male mice (WT). Values are presented as the mean ± SE per milligram protein. In situ hybridization was performed using unilateral orchiectomized animals from wild-type (B and C) and line F (D and E). Endogenous mEP17 expression was detected using an mEP17 antisense probe in intact side (B) and orchiectomized side (C). Epididymis from line F was hybridized with CAT antisense probe. High levels of CAT mRNA were detected in the initial segment of the intact side (D) but no signal in the orchiectomized side (E). Tissues were exposed to emulsion for 1 wk. B–E, Dark field.

 
Ontogeny of 5.3mEP17-CAT transgene expression
To confirm whether the 5.3mEP17 fragment could also be responsible for transgene expression in neonatal mice, transgene expression was studied during postnatal development. In wild-type mice, two mEP17 mRNA transcripts (3.1 and 1.0 kb) were detected in 3-wk epididymis and reached a plateau at 4 wk (Fig. 5AGo). In transgenic mice, no CAT expression was detectable from 1–3 wk of age by CAT assay (Fig. 5BGo). A weak CAT activity was first detected at 4 wk and continued to increase until 6 wk. Although the CAT enzyme assay could not detect activity in 3-wk epididymis, in situ hybridization revealed that the CAT gene transcription was already initiated (Fig. 5DGo), as is the endogenous mEP17 mRNA in the epididymis of the wild-type mouse (Fig. 5CGo). At 3 wk of age, the initial segment is not yet differentiated and cannot be recognized by its histological features, as in the adult. However, in the wild-type, the hybridization signal is restricted to epididymal tubules adjacent to the efferent ducts; whereas in line F, the hybridization signal encompasses a larger area of the caput epididymidis. Furthermore, to examine the regionalization of the transgene expression during epididymal development, the epididymis at various ages were used for in situ hybridization. From 1–2 wk, the initial segment is not differentiated yet, and the epithelium does not express the CAT gene (Fig. 6Go, A and B). At 3 wk, the initial segment begins differentiation, and very few cells express weak CAT mRNA (Fig. 6CGo). At 4 wk, the initial segment is differentiated, characterized by its tall columnar epithelium, and the hybridization signal and cell number of the CAT mRNAexpressing cells are increased and reach a plateau at 5 wk (Fig. 6Go, D–G). These results demonstrate that the onset of the postnatal expression of the transgene also mimics that of the endogenous mEP17 gene.



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  		Figure 5. Ontogenic expression of the endogenous mEP17 and 5.3mEP17-CAT transgene. A, Endogenous mEP17 mRNA was hybridized with 32P-labeled mEP17 cDNA. RNA quality and loading amount was confirmed by ß-actin cDNA. B, Ontogenic 5.3mEP17-CAT expression. CAT activity was determined in epididymides from 1-, 2-, 3-, 4-, 5-, 6-, and 8-wk heterozygous male mice (line F) and compared with that of intact wild-type adult male mice (WT). Three epididymides of 5- to 8-wk mice or 5–9 epididymides of 1- to 4-wk mice were used for CAT assay. Values are presented as the mean ± SE per milligram of protein. *, P < 0.05. Endogenous mEP17 expression was detected in the epididymis, using 35S-labeled mEP17 antisense probe. CAT mRNA was hybridized with 35S-labeled CAT antisense probe in the epididymis of line F mouse. Sections were exposed to emulsion for 7 d. C and D, Dark field; bar, 0.5 mm.

 


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  		Figure 6. Regionalization of the 5.3mEP17-CAT transgene expression during epididymis development. CAT mRNA was hybridized with 35S-labeled CAT antisense probe in epididymides from 1-, 2-, 3-, 4-, 5-, 6-, and 7-wk heterozygous male mice [line F (A–G)]. Sections were exposed to emulsion for 7 d. IS, Initial segment; DC, distal caput; A–G, phase contrast; bars, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tissue-, region-, and cell-specific expression of the 5.3mEP17-CAT transgene
In this study, we examined the 5'-flanking region of the mEP17 gene to investigate the cis-DNA regulatory element involved in the epididymis-specific and testicular factor(s)-dependent gene expression. The 5.3mEP17 fragment corresponding to the 5'-flanking region of the mEP17 gene was isolated from the BAC clone 10983, and ligated to the CAT reporter gene to monitor transgene expression by CAT assay or in situ hybridization. We showed that three of seven transgenic mouse lines displayed a caput epididymidis-specific CAT expression. CAT reporter gene expression was detectable in transgenic mouse line B carrying six copies of the transgene. However, three lines carrying one or two transgene copies showed no CAT activity, suggesting that CAT activity was under the detectable range. On the other hand, line G carries 18 transgene copies, but there was no CAT expression, suggesting that the transgene may have been silenced during transmission into the genome. Therefore, there was no correlation between transgene copy number and transgene expression, suggesting that the integration site may modulate transgene expression levels. In addition, our data showed that transgene expression was weaker than that of the endogenous mEP17 gene. This suggests that the environment surrounding the endogenous gene may be required to reach high expression levels. High expression levels may be conferred by strong tissue-specific enhancers such as locus control regions or matrix attachment regions (MARs). The locus control regions bind to multiple transcription factors that cooperate synergistically to activate gene transcription. On the other hand, the MARs may act as specific landmarks that anchor DNA to the nuclear scaffold (28). It has been shown that a MAR, present on the ovine ß-lactoglobulin gene, modulates basal transgene expression in transgenic mice (29).

The mEP17 last 3' exon is located 1.7 kb from the transcription initiation site of the mE-RABP gene that is also expressed specifically in the epididymis, although restricted to the mid/distal caput epididymidis. This raises the question of whether these genes may share common regulatory elements required for tissue-specific expression. We previously demonstrated that 5 kb of the 5'-flanking region of the mE-RABP gene targets foreign gene expression to the mid/distal caput epididymidis. Interestingly, in the present study, we demonstrate that the 5.3mEP17 fragment also contains all the information for the tissue- and region-specific expression of the endogenous gene, suggesting that the mE-RABP and mEP17 genes have distinct cis-DNA regulatory elements involved in the region-specific gene expression, but they may share elements that are involved in the tissue-specific gene expression.

The 5.3mEP17-CAT transgene expression is regulated by testicular factor(s)
Our study demonstrated that the 5.3mEP17-CAT transgene expression was developmentally regulated and mimicked that of the endogenous mEP17 gene. The 5.3mEP17-CAT transgene expression was correlated with the increase of dihydrotestosterone and androgen receptor content that occurs in the mouse epididymis during postnatal development (30, 31). However, orchiectomy experiments revealed that the 5.3mEP17-CAT transgene is regulated by testicular factor(s) contained in the testicular fluid, not by circulating androgen. The delayed expression of the mEP17 gene and 5.3mEP17-CAT transgene at 3 wk is in agreement with the fact that the efferent ducts become patent only between 15 and 20 d of age. It should be noted that at 3 wk of age, the 5.3mEP17-CAT transgene seems to be less restricted in its expression than the endogenous mEP17 gene in the wild-type. Regionalization of expression of some genes is established progressively during development (32). Although it does not seem to be the case for the endogenous mEP17 gene, the 5.3mEP17-CAT transgene is expressed at first in a wider area of the caput and becomes restricted to the initial segment at 4 wk of age, suggesting that the fragment does not contain all the sequence information contained in the endogenous gene.

Little is known about testicular factors that regulate gene expression in the epididymis. An unidentified sperm-associated factor has been involved in the regulation of the proenkephalin gene in the initial segment of the caput epididymidis (5). In addition, soluble testicular factors, such as growth factors, have also been proposed. The basic fibroblast growth factor has been involved in the regulation of the GGT (33). The basic fibroblast growth factor may act on a MAPK pathway to activate the PEA3 transcription factor that, in turn, may modulate GGT gene transcription. The identification of the cis-DNA regulatory elements that modulate the 5.3mEP17-CAT transgene expression should lead to the identification of new signaling pathways involved in the regulation of epididymal gene expression.

The 5.3mEP17 fragment is useful to target transgene expression in the initial segment
The epididymis has a critical role in gamete biology in promoting the posttesticular development of sperm motility and fertilizing capacity and maintenance of sperm viability. In vitro studies, using epididymal tubules in organ cultures (34, 35) and epididymal cells-spermatozoa cocultures (36), have shown that paracrine interactions with androgendependent secretory proteins are required for sperm maturation and maintenance of sperm viability. The epididymis secretes proteins in a highly regulated and regionalized manner, so that spermatozoa, as they move down the epididymis, encounter a sequence of different luminal fluid environments. Although numerous epididymal proteins have been identified (3), few studies have assigned a biological function to these proteins. The identification of promoters able to target gene expression to the epididymis is not only relevant to identify cis-DNA regulatory elements involved in tissue-, region-, and cell-specific expression of endogenous genes but also to investigate molecular mechanisms involved in epididymal function. Indeed, the ability to overexpress, in specific cells of the epididymis, a gene of interest or foreign genes able to disrupt the function encoded by a gene would be a powerful tool to study epididymal function.

Although epididymal expression of foreign genes driven by promoters of a number of genes expressed in many tissues has been reported, their expression is not restricted solely to the epididymis (3). To our knowledge, the promoters of four genes expressed predominantly or exclusively in the epididymis have been used to target a reporter gene in a specific epididymal segment. The CRISP1 protein is a major component of the murine caudal fluid (12). The murine CRISP1 gene is expressed at high level in the corpus and cauda epididymidis and at low level in the vas deferens and the male submaxillary gland (37, 38). However, a 3.8-kb DNA fragment of the 5'-flanking region of the CRISP1 gene linked to the enhanced green fluorescent protein (EGFP) reporter gene could not direct transgene expression to the epididymis where CRISP1 is normally expressed, but directed expression to the testis where CRISP1 is not normally expressed (39). This suggests that the construct does not contain sufficient information required for epididymis-specific targeting (40). This information could be at the 5' end, in an intron, or at the 3' end. The promoter region of GPX5 has also been used to target EGFP to the epididymis. The endogenous GPX5 gene is expressed at low levels in most mouse tissues but at higher levels all through the caput epididymidis. The 5-kb promoter fragment of the GPX5 gene linked to the EGFP gene directed expression of EGFP not only to a more restricted segment of the caput that was seen with the endogenous gene but also in the cauda and other mouse tissues, albeit at low levels (39). This indicates that the 5-kb promoter fragment of the GPX5 gene does not contain all the regulatory elements necessary to mimic expression of the endogenous gene, but must contain the regulatory elements needed for gene expression in a subsegment of the caput epididymidis. Further, the widespread expression of the transgene limits its usefulness to express genes of interest specifically to the epididymis. Similarly, the long terminal repeats of the mouse mammary tumor virus allow expression of a transgene in the cauda epididymidis but also in other mouse tissues (41). Other promoter regions belonging to two other genes expressed in the caput epididymidis have been evaluated for their ability to target gene transgene expression. The Pem gene encodes a homeobox protein expressed in the principal cells of the caput epididymidis and in Sertoli cells (42). The first 600 bp of the proximal promoter of the Pem gene drives transgene expression in the caput epididymidis but also in Sertoli cells. Similarly, in transgenic mice, the flanking regions of the proenkephalin gene direct expression not only in the initial segment of the caput epididymis but also in the developing germinal cells (43).

In contrast, the 5-kb DNA fragment of the mE-RABP gene promoter targets a foreign gene specifically to the same region of the epididymis where the endogenous mE-RABP gene is expressed and in no other tissues (22). Similarly, as shown in this study, it is possible to introduce a foreign gene in the initial segment in a specific manner using the 5.3 kb 5'-flanking region of the mEP17 gene. As was shown for the mE-RABP transgene (22), the mEP17 transgene displays the same regulation as the endogenous gene. It will now be possible to further dissect and identify the cis-DNA regulatory elements of the mE-RABP and mEP17 genes and their associated proteins (receptor coactivators, transcription factors) that are involved in specific gene expression in the distal caput or the initial segment. In addition, the identification of two epididymis-specific promoters targeting genes to specific epididymal segments, namely the initial segment and the distal caput, will enable one to study the function of the epididymal secretory proteins that spermatozoa encounter as they transit through the epididymis and gain the ability to fertilize.


   Acknowledgments
 
We gratefully acknowledge the Vanderbilt Transgenic/ES Cell Shared Resource of the Vanderbilt-Ingram Cancer Center (NCI Grant 2P30-CA-68485) for generating the transgenic mouse lines and the DNA Sequencing Core of the Vanderbilt-Ingram Cancer Center for DNA sequencing.


   Footnotes
 
This work was supported by NIH Grant HD-36900 and a grant from the Rockefeller Foundation/Ernst Schering Foundation.

The nucleotide sequence reported in this paper has been submitted to the GenBank with GenBank accession no. AF082221.

1 Present address: Department of Immunology and Parasitology, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan. Back

Abbreviations: CAT, Chloramphenicol acetyltransferase; CRISP, cysteine-rich secretory protein; EGFP, enhanced green fluorescent protein; GGT, {gamma}-glutamyl transpeptidase; MAR, matrix attachment region; ß-ME, ß-mercaptoethanol; mEP17, murine epididymal protein of 17 kDa; mE-RABP, murine epididymal retinoic-acid-binding protein; SSC, saline sodium citrate.

Received July 24, 2002.

Accepted for publication November 22, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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