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Departments of Obstetrics and Gynecology (S.F., K.S., M.-C.O.-C.), Biochemistry (P.C., B.B.D., D.E.O., R.M.C.), Cell Biology (G.E.O., M.-C.O.-C.), Urologic Surgery (R.J.M.), Mass Spectrometry Research Center (P.C., B.B.D., R.M.C.), and Center for Reproductive Biology Research (S.F., K.S., D.E.O., G.E.O., R.J.M., M.-C.O.-C.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and Institut National de la Recherche Agronomique, Station Commune de Recherche en Ichtyophysiologie, Biodiversité et Environnement (J.-J.L.), Campus de Beaulieu, F-35042 Rennes Cedex, France
Address all correspondence and requests for reprints to: M.-C. Orgebin-Crist, Ph.D., Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Room C-3306 MCN, Nashville, Tennessee 37232-2633. E-mail: m-c.orgebin-crist{at}mcmail.vanderbilt.edu.
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During ontogeny, mEP17 was detected as early as 3 wk of age and increased afterward. After bilateral orchiectomy, mEP17 disappeared 2 d after surgery and was not restored after testosterone replacement. After unilateral orchiectomy, mEP17 levels decreased only in the orchiectomized side. After cryptorchidism or busulfan treatment, mEP17 levels were either greatly diminished or not detected. This suggests that mEP17 is dependent on testicular factor(s) that may have a germ cell origin.
Altogether, our data demonstrate that mEP17 spatial expression, regulation, and fate are different from that of the highly related mouse epididymal retinoic acid binding protein. This suggests that these two related proteins exhibit distinct functions in the mouse epididymis.
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Introduction |
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Some secreted epididymal proteins belong to the lipocalin superfamily. Lipocalins include a large group of low-molecular-weight secreted proteins conserved during evolution from prokaryotes to mammals. They share a highly conserved three-dimensional (3-D) structure that contrasts with a low level of primary sequence similarity (5, 6, 7). This specific 3-D structure delimits an internal hydrophobic pocket involved in the binding of small lipophilic molecules (8, 9). However, despite their common structure, lipocalins present diverse ligand specificity and selectivity. They are involved in transport of various lipophilic molecules, such as a fatty acid for protooncogene 24p3 (10), retinol for plasma retinol binding protein (11), and cholesterol and steroid hormones for apolipoprotein D (12). Consequently, lipocalins display large and diverse physiopathological effects.
In mammals, lipocalins are ubiquitous, present in various fluids, like urine, tears, saliva, and cerebrospinal fluid. They have been shown to be present in the fluids of both male and female genital tracts in several species. In the mouse epididymis, four lipocalins have been identified to date: prostaglandin D2 synthase (PGDS), also present in the testis (13, 14); protein 24p3 (15), also present in uterine fluid; and two genes specifically expressed in the epididymis, mE-RABP (mouse epididymal retinoic acid binding protein) (16) and mEP17 (mouse epididymal protein of 17 kDa) (18). The biological significance of lipocalins in the epididymis is uncertain; however, several studies suggest that this family might be important for sperm maturation and/or survival. For example, E-RABP, the 3-D structure of which was resolved by x-ray crystallography (19), may be involved in retinoid trafficking (20, 21), an important signaling pathway controlling epididymal function (22). PGDS was shown to be a biological marker of sperm quality in humans (23) and to bind retinoids and androgens in vitro (24, 25). There are several putative lipophilic ligands for lipocalins in the epididymis. Moreover, there are various other proteins that can ensure the binding of lipophilic molecules in the epididymal fluid, such as human epididymal 1/cholesterol transfer protein for cholesterol (26), and androgen-binding protein (ABP) for 5
-dihydrotestosterone (27).
The lipocalins mE-RABP and mEP17 genes evolved by gene duplication from an ancestral gene (18). To date, they are the only lipocalins specifically expressed in the epididymis. According to our previous studies, mE-RABP and mEP17 mRNA show a highly regionalized and different pattern of gene expression in the epididymis. These related genes constitute a unique model to better understand the molecular mechanisms involved in the regionalization and regulation of gene expression in the epididymis.
To demonstrate whether the mEP17 transcripts are translated in vivo, a specific antibody was raised against a recombinant mEP17 (RmEP17). The temporal and spatial expression of mEP17 was studied in the epididymis during postnatal development, and its regulation by testicular factors was investigated. Our data demonstrate that mEP17 and mE-RABP genes display not only a different region-specific expression pattern and regulation but also a different fate of their encoded proteins.
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Materials and Methods |
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This study was performed on ICR/CD-1 mice and Sprague Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN). Animals were killed by cervical dislocation after anesthesia under methoxyflurane (Mallinckrodt, Inc., Mundelein, IL). The epididymis and testis were removed after abdominal incision, and the epididymis was dissected from the testis before freezing and storage at -80 C before protein extraction. When required, the epididymis was separated into four regions: initial segment (segment 1), caput (segments 2–4), corpus (segments 5 and 6), and cauda (segments 7 and 8), epididymal segments being numbered according to the classification of Soranzo et al. (28). For immunohistochemistry, the whole epididymis was fixed in Bouin’s fluid (VWR Scientific Products, Atlanta, GA) for 48 h.
The age of the adult animals was 8–10 wk for the mice and 2 months for the rat. For the postnatal development study, mice were killed at 1, 2, 3, and 4 wk of age, with a pool of at least six pups for each time point.
For the regulation analysis, bilateral orchiectomy and unilateral orchiectomy were performed by the abdominal route, under light anesthesia, on 9-wk-old mice. Animals were killed 2, 3, 6, 8, and 10 d after orchiectomy. For each time point, tissues from four mice were pooled for protein extraction. The effect of circulating androgens was analyzed by testosterone replacement in orchiectomized mice. Injections (im) of 2.5 mg/kg testosterone propionate (Sigma, St. Louis, MO) were given daily for 10 d, beginning 3, 6, 8, and 10 d after orchiectomy. The seminal vesicle weight of all animals was recorded. After unilateral orchiectomy, mice were killed 2, 3, and 7 d after surgery. For each time point, the epididymal tissues from the nonorchiectomized side of four mice were pooled for protein extraction, and the same was done with epididymal tissues from the orchiectomized side.
The effect of cryptorchidism was analyzed in four mice. For each mouse, one testis/epididymis was maintained in the abdominal cavity during 30 d, by ligature of the epididymal fat on the internal abdominal skin. The other side served as control. Busulfan treatment was performed, as described by de Rooij and Kramer (29), by a single ip injection of 10 mg/kg. The busulfan was first dissolved in acetone and then in sesame oil. Control mice received the vehicle alone. The treated mice were killed 35 and 55 d after the injection. After cryptorchism and busulfan treatment, testes were weighted and fixed in Bouin’s fluid for histological assessment of spermatogenesis.
Production of RmEP17/His-Tag and mE-RABP/His-Tag
Construction of the expression vector.
The full-length cDNA encoding mEP17 was previously ligated into the pGEM-T cloning vector (Promega Corp., Madison, WI) to generate constructs F2 and F3 (18). The open reading frame encoding mature mEP17 protein was amplified by PCR using slmEP17 (5'ACCATGGCTGAGTCTACCAGGGTGGAGCTT-3') and aslmEP17 (5'-CTGCAGACCAGTCCCTCTTTCAAGAGCTA-3') as primers and construct F2 as template. Primers s2mE-RABP (5'-CTCGAGACAGAGGCTGCAGTGGTGAAGGATTTC-3') and as2mE-RABP (5'-GGTACCAGATCTGTCCCGATTGCAATGC-3') and the E7 construct (16) were used to amplify the open reading frame corresponding to the minor form of mE-RABP. Ncol and Pstl restriction sites were included at the 5' end of slmEP17 and aslmEP17 primers, respectively, to facilitate subsequent subcloning. Similarly, Xho1 and Kpn1 restriction sites were included at the 5' end of s2mE-RABP and as2mE-RABP primers. Twenty nanograms of plasmid DNA F2 or E7 were mixed with PCR buffer II (Perkin-Elmer Corp., Foster City, CA), 2 U Taq B DNA polymerase (Promega Corp.), 1.5 mM MgCl2, 1 µM concentration of each primer (slmEP17 and aslmEP17), and 0.2 mM deoxynucleotide triphosphate. The 500-bp DNA fragment corresponding to the mEP17 and mE-RABP open reading frame was amplified for 30 cycles (95 C, 1 min; 50 C, 45 sec; 72 C, 45 sec) followed by one cycle (95 C, 1 min; 50 C, 45 sec; 72 C, 10 min) using a GeneAMP 2400 thermocycler (PE Applied Biosystems, Courtaboeuf, France). The PCR products were purified on a 1.5% (wt/vol) agarose gel and cloned into the pCR2.1 cloning vector using the TOPO-TA Cloning System (Invitrogen, Groningen, The Netherlands) to generate construct pCRF2 and pCRE7. The open reading frame encoding mEP17 and mE-RABP were produced from the pCRF2 and pCRE7 constructs digested with Ncol and Pstl endonucleases. The DNA fragment Ncol/Pstl was purified on a 1% (wt/vol) agarose gel and ligated into the pBAD/gIIIa bacterial expression vector (Invitrogen), previously linearized using the Ncol and Pstl restriction enzymes. Ligation product was transformed in DH5F' and grown on a Luria-Bertani (LB)/100 µg/ml ampicillin (Amp) agar plate at 37 C. Finally, the construct (pBADF2) was purified from recombinant colonies and sequenced to check that the open reading frame encoding the mature mEP17 was still in frame with the N-terminal secretion signal and the C-terminal 6xHis tag. Sequencing DNA reactions were performed according to manufacturer’s instructions (PE Applied Biosystems) using pCR2.1 vector primers. DNA fragments were separated and analyzed using the automated ABI 310 sequencer (PE Applied Biosystems). Nucleotide sequences were analyzed using Seqanalysis and Autoassembler softwares (PE Applied Biosystems).
Expression of mEP17/His-Tag and mE-RABP/His-Tag.
TOP 10 competent cells (Invitrogen) were transformed using 10 ng pBADF2 or pBADE7 constructs and grown on LB/50 µg/ml Amp agar plate at 37 C. Single recombinant colonies from the plate were inoculated into 2 ml LB/Amp medium. The growth of the selected colonies was carried out according to the instruction manual "pBAD/gIII A, B, and C Vectors for Regulated, Secreted Expression of Recombinant Proteins Containing C-Terminal 6xHis Tags in E. coli" (Invitrogen). Briefly, cells were grown at 37 C overnight at 250 rpm. A 0.1-ml vol of the overnight culture was used to seed 10 ml LB/Amp medium and allowed to grow at 37 C until an A600 of 0.5 was reached (
2 h). Expression was then induced by the addition of L-arabinose to a final concentration of 0.02%. After incubation for 4 h at 37 C and 250 rpm, cells were centrifuged, and the pellet was resuspended either in 100 µl 1x SDS-PAGE sample buffer for direct electrophoretic analysis, or in 2.5 ml lysis buffer (50 mM NaH2PO4, 10 mM imidazole, 300 mM NaCl). In this case, after 30 min on ice, cells were sonicated for 5–10 min on ice at maximum power. After centrifugation at 7500 x g for 20 min at 4 C, the pellet was resuspended in 5 ml lysis buffer, and both supernatant and pellet were analyzed on Western blot using the His-Tag antibody linked to peroxidase (Invitrogen). The RmEP17 sample was used as antigen for antibody production after 1-D electrophoresis and a light Coomassie blue staining (Amersham Pharmacia Biotech, Piscataway, NJ) as described below.
IgG production using the recombinant protein in the gel
After one-dimensional (1-D) electrophoresis, the bands corresponding to the RmEP17 were excised from 16 gels and pooled. After homogenization in 5 ml 1x PBS, four aliquots of 1 ml were prepared. The first aliquot was mixed with 1 ml complete Freund’s adjuvant (Sigma), and 1 ml of the emulsion was intradermally injected into two rabbits on their back (10 injections of 0.1 ml). Three following injections were performed at 10-d intervals, using incomplete Freund’s adjuvant. Ten days after the last injection, the rabbits were killed, and the serum was used for IgG purification using a Protein A column kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions.
cDNA cloning of the full-length mEP17 and in vitro transcription/translation assay
The full-length cDNA sequence of mEP17 (clones F2 and F3) previously constructed by Lareyre et al. (18) was used for in vitro transcription/translation assay using the TNT/reticulocyte coupled system from Promega Corp. in the presence of (35S)-methionine, according to the manufacturer’s instructions. Sense and antisense RNA were produced from the F3 clone using T7 and SP6 RNA polymerase, respectively. Thirty microliters of the sense RNA translation reaction were subjected to immunoprecipitation using Pansorbin reagent (Calbiochem-Novabiochem Corporation, San Diego, CA) as described previously (29), with 20 µg mEP17 IgG or 20 µg normal rabbit IgG (DAKO Corp., Capinteria, CA) as negative control. Samples were analyzed by 1-D SDS-PAGE followed by fluorography using X-OMAT AR films (Eastman Kodak Co., Rochester, NY).
1-D and two-dimensional (2-D) gel electrophoresis
Proteins from whole epididymis, as well as initial segment, caput, corpus, and cauda, were extracted by homogenization in 50 mM Tris-HCl (pH 7.4), 0.5 mM EDTA at pH 8, and 2% vol/vol of a protease inhibitors cocktail (Sigma). The tissue homogenates were centrifuged twice at 15,000 x g for 30 min at 4 C, and the concentration of total proteins was estimated by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). SDS-PAGE separation was carried out, according to the method of Laemmli (31), on 12% polyacrylamide gels. Then, 1-D and 2-D electrophoresis was performed with 15 µg and 180 µg total proteins, respectively. For 2-D electrophoresis, isoelectric focusing was performed using pH 3–10 IPG strips (Bio-Rad Laboratories, Inc.). Gels were stained with Coomassie blue or Sypro Ruby (Bio-Rad Laboratories, Inc.), depending on the experiment, or used for electrotransfer for Western blotting analysis as described below.
Detection of EP17 and E-RABP by Western blotting and immunohistochemistry
The mE-RABP antibody was obtained, characterized, and used as described previously (32). Western blotting analyses were performed after electrophoresis separation and subsequent electrotransfer of the proteins onto a polyvinyldifluoride membrane (Millipore Corp., Bedford, MA) in 25 mM Tris/190 mM glycine/20% methanol buffer. After incubation in the blocking buffer [3% BSA (Sigma) in 1x PBS-0.5% Tween], the membranes were incubated overnight at 4 C with the immune IgG diluted at 1 µg/ml or nonimmune rabbit IgG (DAKO Corp.) at the same concentration. After three washes, the blots were incubated with horseradish peroxidase-conjugated goat antirabbit IgG (DAKO Corp.) and then washed again. Immunoreactive proteins were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
For immunohistochemistry, freshly dissected tissues were fixed in Bouin’s fluid for 24 h, washed in ethanol 50%, then dehydrated before embedding. Immunodetection was performed on 5-µm sections. Endogenous peroxidase activity was quenched with 0.03% hydrogen peroxide in sodium azide for 30 min, and nonspecific binding sites were blocked in the blocking buffer (DAKO Corp.). Tissue sections were exposed to the antibody diluted at 10 µg/ml or to normal rabbit IgG (DAKO Corp.) at the same concentration at 4 C overnight. After incubation with the secondary antibody (DAKO Corp.) and washes, immunostaining was performed with diaminobenzidine substrate-chromogen (DAKO Corp.) according to manufacturer’s instructions.
Immunofluorescence
Cells were fixed for 45 min on ice with 4% formaldehyde in 0.1 M sodium phosphate, pH 7.4. After adhering to poly-L-lysine-coated coverslips, cells were rinsed in TBS [150 mM NaCl, 20 mM Tris HCl (pH 8.0), 0.05% Tween 20] and blocked with TBS containing 5% goat serum and 2.5% BSA. Coverslips were then incubated in primary antibody (purified IgG-diluted in blocking solution, 5 µg/ml); controls received equivalent dilutions of nonimmune IgG. After three washes in TBS-1% BSA, the coverglasses were incubated in TBS-BSA containing affinity-purified secondary antibodies of Cy3-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Coverslips were then rinsed several times in TBS, and cells were examined by phase contrast and epifluorescence microscopy.
N-glycanase digestion
N-glycanase digestion was performed using the recombinant N-glycanase (Glyko, Inc., Novato, CA) according to manufacturer’s instructions. Fifty micrograms of total epididymal initial segment proteins in a final vol of 50 µl were digested at 37 C for 0, 18, and 36 h. An aliquot of 15 µl was removed at each time point and analyzed by Western blotting.
Preparation of EP17-enriched HPLC fractions from mouse epididymis initial segment and caput postmicrosomal supernatants
Frozen tissues, dissected from 11 mice, were thawed, minced with dissection scissors, and homogenized, by hand, in a glass-glass Duall homogenizer (0.1–0.15 mm clearance; Kontes Glass Co., Vineland, NJ) at 4 C until microscopic examination indicated adequate tissue disruption. The homogenization medium was prepared by combining 2.5 ml 1.0 M sucrose, 1.0 ml 0.1 M Tris-HCl (pH 7.4 at room temperature), 0.1 ml 10 mM phenylmethylsulfonyl fluoride (in isopropanol), and 6.4 ml water (final pH of 7.6 at 4 C). The ratio of homogenization medium to pooled tissue was 8 to 1 (vol/wt) for the initial segment and approximately 10 to 1 for the caput. The homogenates were fractionated by differential centrifugation at 4 C to produce a crude nuclear pellet (680 x g, 10 min), crude mitochondrial/lysosomal pellet (10,000 x g, 10 min.), and a postmicrosomal supernatant and microsomal pellet (55,000 rpm, 60 min; TLA-100.3 fixed angle rotor, TL Optima Ultracentrifuge; Beckman, Fullerton, CA).
Chromatography was carried out on an Alliance HPLC system (Waters Corp., Milford, MA) configured with a 2690 separations module and a 2487 dual-wavelength absorbance detector and computer controlled with Millenium (32) software (version 3.05.01). Postmicrosomal supernatants were fractionated on a Vydac (Hesperia, CA) 259VHP5415 polymeric column (5-µm particle, 4.6 mm x 15 cm) at 40 C. Eluents A and B were, respectively, 1000/1 (vol/vol) water/trifluoroacetic acid (TFA), and 1000/0.85 (vol/vol) acetonitrile/TFA. Two 100-µl portions of postmicrosomal supernatant were loaded (separated by a 5-min hold at 5% B) before the solvent program was initiated. The solvent program was as follows: 5 min hold at 5% B; linear gradient from 5–60% B over 55 min; linear gradient from 60–95% B over 5 min; 5 min hold at 95% B; linear reverse gradient to 5% B over 5 min. Proteins were eluted at 1 ml/min flow rate, and 1-min fractions were collected into cold Eppendorf (Brinkmann Instruments, Inc., Westbury, NY) Safe-lock microcentrifuge tubes. Fractions were frozen and lyophilized dry. Each fraction was then reconstituted [in 10 µl 1000/1 (vol/vol) water/TFA] and analyzed by dot-blotting using the mEP17 antibody. Two microliters of each fraction were deposited on a polyvinyldifluoride membrane and further analyzed as described above for Western blotting. The reactive fractions in initial segment and caput were then pooled and analyzed by Western-blotting and stained electrophoresis.
Trypsin in-gel digestion for mass spectrometry (MS) analysis
After electrophoresis and staining with Coomassie blue (bacterial lysates) or Sypro Ruby (tissue lysates), bands to be analyzed by MS were excised, then washed in 30% methanol and in 100 mM ammonium bicarbonate. After crushing the gel into small pieces, the proteins in the gel were reduced with 10 mM dithiothreitol (Sigma) at 60 C and alkylated with 10 mM iodoacetamide (Sigma), at room temperature, in the dark. The gel pieces were then washed in 50 µl acetonitrile and subsequently dried under vacuum. In-gel trypsin digestion was performed overnight at 37 C in 25 µl 50-mM ammonium bicarbonate with 0.02 µg/µl of sequencing grade modified trypsin (Promega Corp.).
Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS
MALDI MS analyses were performed on a Voyager MALDI DE-STR mass spectrometer (PE Applied Biosystems, Framingham, MA). Protein profiling of the HPLC fractions was performed by MALDI MS in the linear acquisition mode under optimized delayed extraction conditions using sinapinic acid as matrix (prepared at 10 mg/ml in 50/50/0.1 acetonitrile/H2O/TFA by volume). One microliter of the reconstituted HPLC fractions was mixed on target with 1 µl matrix. The mixture was allowed to dry at ambient room temperature before mass analysis. Peptide mapping of the digestion products obtained after in-gel digestion of the RmEP17 was performed by MALDI MS in the linear acquisition mode under optimized delayed extraction conditions using -cyano-4-hydroxy-cinnamic acid as matrix (prepared at 10 mg/ml in 50/50/0.1 acetonitrile/H2O/TFA by volume). After in-gel digestion, 1 µl of the supernatant was mixed on target with 1 µl matrix. The mixture was allowed to dry at room temperature and was then analyzed.
Nanoelectrospray (nanoES) quadruple-TOF (QqTOF) MS
NanoES QqTOF MX was performed on an MDS Sciex (Concord, Canada) QStar instrument fitted with a Protana (Odense, Denmark) nanospray source. Positive ion spectra were acquired in the profile mode, with an ion count accumulation time of 1 sec and a TOF pulser frequency of 6.99 kHz. A stable spray was obtained at 800–900 V. Data were acquired in the TOF MS mode with nitrogen curtain and collision gas settings of 15–20 
and 3–4 (arbitrary units), respectively. Peptide sequences were obtained from product ion spectra by tandem MS (MS/MS) acquired for ions selected with Q1 and dissociated by increasing the collision gas setting and Q0 offset potential.
NanoES QqTOF MS/MS spectra of peptides from the in-gel trypsin digest of reduced and carboxyamidated RmEP17 were obtained after simply diluting 1 µl of the digest with 9 µl 60/39/1 (vol/vo/vol) methanol/water/88% formic acid. The trypsin digests (30 µl) of the main gel band (and its upper and lower margins), obtained for the reduced and carboxyamidated pooled HPLC fractions 43 derived from tissue postmicrosomal supernatants, were acidified by the addition of 3 µl 8.8% formic acid and desalted on a C18 Zip-tip (Millipore Corp.). The Zip-tip was washed with 0.18% formic acid before adsorbed peptides were eluted from the tip in 3–4 µl 70/29/8/0.2 (vol/vol/vol) methanol/water/88% formic acid.
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). Therefore, the expected molecular mass for the proform of RmEP17 was 24 kDa. However, cleavage of the bacterial signal sequence that replaces the endogenous EP17 signal peptide would result in the loss of 2 kDa and lead to a 22-kDa mature form of RmEP17. The recombinant mE-RABP (RmE-RABP) was produced as well, being generated from the E7 clone (16) and expressed in the same vector (Fig. 1A). For RmEP17, after 1-D electrophoresis of the bacteria pellet proteins and Western blotting analysis using the His-Tag antibody, two bands were detected at 24 and 22 kDa, after induction by arabinose (Fig. 1B). The molecular mass of these bands was in agreement with the theoretical molecular mass of the unprocessed (24-kDa) and processed (22-kDa) forms of RmEP17. For RmE-RABP, a single 23-kDa band was observed corresponding to the unprocessed form (Fig. 1B). These results suggested that the processing of the recombinant proteins was mostly incomplete. After sonication, the two bands of RmEP17 were detected in the pellet (not shown), suggesting that the two forms of the protein were insoluble. The identity of the RmEP17 protein of 24 kDa was first verified by MALDI MS peptide mass mapping after in-gel trypsin digestion of the appropriate gel band (data not shown). Most of the observed peptides were in accordance with the RmEP17 theoretical peptide map, covering over 86% of the sequence (Fig. 1D). To unambiguously identify the protein, several tryptic peptides detected by MALDI MS were sequenced by nanoESI MS/MS. In particular, from the MS/MS spectrum presented in Fig. 1C, obtained from the doubly charged ion at mass to charge ratio 493.79 (molecular weight 985.58), the recovered sequence information was in perfect agreement with the [118–125] RmEP17 tryptic peptide (see fragmentation scheme in Fig. 1C).
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) corresponding to the two bands of RmEP17 recognized by the His-Tag antibody (Fig. 2B). The RmE-RABP, as well as the short peptide generated from the native expression vector, were not detected by the RmEP17 immune purified IgG (Fig. 2A). These results suggested that the purified immune IgG was specific for RmEP17.
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). The higher molecular mass of the in vitro translation product, in comparison with the theoretical molecular mass (26 kDa vs. 19.8), may be the result of posttranslational processes, such as glycosylations, that may occur in the cell-free translation system, as previously observed in previous studies (33). The protein of 26 kDa was specifically immunoprecipitated after incubation in the presence of the immune IgG but not in the presence of normal rabbit IgG (Fig. 3B).
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By Western blotting analysis, a 22-kDa band was observed in the initial segment and caput epididymidis in both mouse and rat (Fig. 4, A and B); whereas no signal was obtained with the preimmune serum or normal rabbit IgG (not shown). In both species, the 22-kDa antigen was present at higher levels in the initial segment than in the caput epididymidis. No protein was detected in mouse and rat testis, corpus, and cauda epididymidis, as well as in various male and female murine tissues, including organs of the genital tract (Fig. 4C).
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, A and B) and in the clear cells, apical cells, and lumen of the midcaput (Fig. 5, A and C). As a negative control, no signal was observed using normal IgG (Fig. 5D). To determine whether mEP17 is a sperm-binding protein, caput and cauda epididymal spermatozoa were immunostained with the RmEP17 antibody. No immunofluorescence was observed on formaldehyde-fixed spermatozoa from either the caput or the cauda epididymidis (data not shown). Coupled with the result that mEP17 mRNA is localized in the initial segment (18), these results strongly suggest that the protein synthesized in the initial segment is secreted and accumulates in the lumen of the mid/distal caput but does not bind to spermatozoa.
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, A and C. Immunoreactivity for mEP17 was observed for both separations in the 43-min HPLC fractions (Fig. 6, B and D). When monitoring these fractions by MALDI MS, no strong signals in the 21- to 23-kDa range potentially corresponding to mEP17 were observed (not shown). Furthermore, from the MALDI MS analysis, it was estimated that both fractions contained high amounts of -hemoglobin and mouse serum albumin. From the intensity of the UV profiles, a total protein amount of about 80 µg was estimated per 43-min fractions. To further separate mEP17 from these and other abundant proteins, the two 43-min HPLC fractions were pooled and separated by 1-D gel electrophoresis (Fig. 6E). Two gel lanes were loaded with approximately 80 and 16 µg for subsequent MS and Western blot analysis, respectively. A single band corresponding to mEP17 was identified on Western blot. In the adjacent lane, a gel slice corresponding to the molecular-weight position of mEP17 was excised and subdivided in three smaller slices for trypsin digestion (Fig. 6E). After digestion, the resulting tryptic peptides were analyzed by nanoES MS/MS. Three mEP17 tryptic peptides covering over 22% of the total protein sequence were detected and successfully sequenced (Fig. 1D
). Figure 6F presents the MS/MS spectrum and resulting fragmentation scheme of the [118–125] mEP17 tryptic peptide obtained from its doubly charged ion at mass to charge ratio 493.79. This MS/MS spectrum is in good agreement with the one obtained for the same peptide from the RmEP17 (data not shown).
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) suggested that the endogenous protein was likely glycosylated. To confirm this hypothesis, N-deglycosylation of the proteins extracted from the initial segment of the mouse epididymis was carried out. As expected, the molecular mass of the 22-kDa antigen decreased to 17 kDa, the theoretical molecular mass of the mature form of mEP17 (Fig. 7A). After 2-D electrophoresis and Western blotting, the native protein was resolved into five isoforms with an isoelectric point (pI) from 5.8–6.7 (Fig. 7B). These spots, after staining with Sypro Ruby, were not visualized (not shown), strongly suggesting that mEP17 was a minor component, as suspected after HPLC purification and MS analysis. The N-deglycosylation gave rise to a single spot at pI 6 (Fig. 7C), the theoretical pI being in the range of 5.7–6.
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) and at 3 wk of age by immunohistochemistry (Fig. 8, C and D). However, the immunostaining was faint, with a checkerboard pattern, and was restricted to the most distal region of the initial segment. At 4 wk, the immunostaining was strong and the cellular localization was similar to that observed in adult mice, with mEP17 localized in the principal cells of the initial segment and lumen of the proximal caput epididymidis (Fig. 8, E and F). mE-RABP expression was clearly detectable by Western blotting in 3-wk-old mice (Fig. 8B), confirming a previous study in which the protein was first detected between 20 and 30 d (34).
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The seminal vesicles weight, expressed in percentage of the body weight, was 0.4%, 0.3%, 0.1%, and 0.1% after 2, 6, 8, and 10 d of orchiectomy, respectively, compared with 0.7% in normal mice of the same age. As expected from what was previously shown at the mRNA level (18), mEP17 expression strongly decreased after orchiectomy, starting 2 d after surgery (Fig. 9A). Testosterone supplementation did not restore the expression of the protein (Fig. 9A). It was previously shown that mE-RABP expression is under the control of circulating androgens (16). In our experiment, mE-RABP level decreased progressively after bilateral orchiectomy, was barely detectable 6 d after castration (Fig. 9B), and was reinduced by testosterone supplementation. These results confirm the previous study (16) showing that mE-RABP decreased by 50% at 5 d after castration and was no longer detected after 10 d.
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, A, C, and D). The level of mE-RABP was similar in the normal and orchiectomized sides (Fig. 10B). Altogether, these results confirm that mEP17 expression is under the control of testicular factors reaching the epididymis through the efferents ducts.
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After 1 month of cryptorchidism, mEP17 expression decreased to a barely detectable level in the cryptorchid side, whereas the intact side showed normal expression of the protein (Fig. 11A). The level of mE-RABP was not affected by cryptorchidism (Fig. 11B). On histological sections of the cryptorchid testis, no spermatozoa and spermatids, fewer spermatocytes, and mostly spermatogonial cells could be observed, suggesting an arrest of spermatogenesis at the meiotic stages (Fig. 11D). In contrast, spermatogenesis in the contralateral testis was normal (Fig. 11C). The weight of the cryptorchid testis was 54% decreased, compared with the intact side.
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). The level of mE-RABP was not affected by the busulfan treatment (Fig. 12C). The testis weight of the busulfan-treated mice was 39% decreased, compared with that of control mice. In the seminiferous tubules, there was a striking depletion of pachytene spermatocytes (Fig. 12E). Fifty-five days after the injection of busulfan, the level of mEP17 was close to normal (Fig. 12B). In the testis, the most conspicuously depleted germ cells were the elongated spermatids in stages I–VIII of Roosen-Runge’s classification of the seminiferous epithelium cycle (34A ), all other germ cells being present (Fig. 12F). Taken together, the data indicate that mEP17 expression is correlated with normal spermatogenesis.
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Discussion |
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Identification of endogenous mEP17
We have previously shown that mEP17 mRNA is specifically expressed in the initial segment of the murine epididymis (18). To demonstrate the presence of the endogenous mEP17, tissue extracts from the initial segment and segments 2 and 3 of the caput were fractionated by HPLC and monitored by MALDI MS and dot blotting using the antibody to RmEP17. Unfortunately, the immunoreactive fractions were contaminated with serum albumin and -hemoglobin. However, these fractions were pooled and separated by 1-D gel electrophoresis, and the immunoreactive band was excised and trypsinized. The resulting tryptic peptides were analyzed by nanoES MS/MS. Three tryptic peptides covering over 22% of the total protein sequence were identified as mEP17. Furthermore, MS/MS spectra obtained from three peptides were in good agreement with the spectra from the corresponding peptides of the RmEP17. MS is a sensitive technique allowing detection and sequencing of proteins in low abundance, and this approach led to the unambiguous identification of endogenous mEP17. The protein may be a minor component in the epididymis, because it was not stained with Sypro Ruby (staining as sensitive as silver staining), in native tissue extract. However, despite its low abundance, the protein was easily detected by the RmEP17 antibody, suggesting that this antibody had a high titer and was highly specific.
EP17 immunolocalization and characterization in the mouse epididymis
Western blot analysis showed that the polyclonal antibody raised against the RmEP17 recognized a 22-kDa protein in the initial segment and proximal caput epididymidis. The expression of this protein in the initial segment is in agreement with that of the mEP17 transcripts (18). mEP17 possesses the sequence characteristics of the lipocalin family (in particular, the conserved motifs involved in the 3-D structure) and the presence of a signal peptide, suggesting that the protein is extracellular. It is thus not surprising that mEP17 was detected in the lumen in the proximal caput epididymidis. However, the molecular mass of the protein was higher than that deduced from the open reading frame of the mEP17 mRNA. This suggested that the endogenous mEP17 could be subjected to posttranslational modifications. Further analysis confirmed this hypothesis, because the apparent molecular mass of the antigen decreased to 17 kDa after N-glycanase digestion. Although the mEP17 amino acid sequence analysis showed two putative N-glycosylation sites, corresponding to asparagine residues at positions 66 and 74, it was not known whether both sites are involved in this process. Glycosylation is a common feature of secretory proteins. We have previously shown that the murine epididymal PGDS, which also belongs to the lipocalin family, is N-glycosylated (14). The biological significance of N-glycosylations of epididymal secretory proteins remains poorly understood; however, such posttranslational modifications could modulate protein functions, as previously demonstrated for the enzymatic activity of the 
-glutamyl transpeptidase in the rat epididymis (36). Although the 2-D gel analysis of the endogenous mEP17 revealed only one spot after N-glycosylation, other posttranslational modifications cannot be excluded. For example, analysis of the amino acid sequence of mEP17 revealed three putative sites of protein kinase C phosphorylation and four putative sites of casein kinase II phosphorylation.
Immunohistochemical studies showed a specific immunostaining of the supranuclear region of the principal cells of the initial segment. This indicates that the protein is synthesized by the principal cells of the initial segment, is then secreted into the lumen of the epididymal duct, but does not seem to bind to spermatozoa. The absence of immunoreactivity in the lumen of the initial segment is likely attributable to the rapid flow of fluid through that particular region of the epididymis. The initial segment, like the efferent ducts, are involved in fluid reabsorption, leading to a concentration of the luminal fluid components in more distal regions. Therefore, it is not surprising that mEP17 cannot be detected in the lumen of the initial segment but is readily detected in the proximal caput epididymidis. The protein was not only detected in the lumen but also in the clear cells of the proximal caput epididymidis. Because mEP17 transcripts were not detected in these cells by in situ hybridization (18), it suggests a reabsorption process of the protein in these particular cells. This reabsorption process, that is probably very active considering the number of immunoreactive cells, is likely responsible for the disappearance of the luminal protein in the more distal part of the epididymis. However, one cannot exclude that a specific degradation of the secreted mEP17 protein occurs also in the lumen of the distal epididymis. Indeed, proteases have been identified in the epididymal fluid (2). In contrast, other abundant epididymal secretory proteins, such as mE-RABP and PGDS, accumulate in the luminal fluid of the cauda epididymidis despite an active reabsorption process in clear cells of the distal corpus and cauda epididymidis (14, 32). Numerous membrane-bound molecules have been proposed as receptor for lipocalins (37), but their localization on clear cells remains unknown. Further investigations will be required to determine the molecular mechanisms leading to the specific reabsorption of epididymal lipocalins by clear cells.
Conservation of EP17 during evolution
In the rat epididymis, the polyclonal antibody raised against mEP17 identified an antigen with an immunolocalization pattern identical with that seen in the mouse. The presence of an EP17-like protein in the rat is in agreement with the Southern blot analysis of genomic DNA showing that a mEP17-like gene is present in the rat genome (18). The molecular mass of the endogenous EP17 (22 kDa) was similar in both species.
Our attempts to demonstrate the presence of EP17 in other species (including hamster, pig, sheep, monkey, and human) were unsuccessful using the polyclonal antibody raised against the recombinant mouse protein (unpublished results). This does not imply that a mEP17-like gene is not present in the genome of these species. Indeed, EP17 mRNA is expressed and localized in the initial segment of the hamster epididymis (18). Moreover, by analysis of the human genome, a human homolog of EP17 has been identified (38). The low amino acid sequence homology commonly observed between orthologous lipocalins may prevent antigen recognition.
mEP17 is regulated by luminal testicular factors
Epididymal expression of some genes depends on testicular factors present in luminal fluid (39, 40, 41, 42). In the present study, mEP17 disappeared from the epididymis after bilateral and unilateral orchiectomy. Testosterone replacement in bilateral orchiectomized mice failed to restore protein expression. These observations are in agreement with the previous report showing that mEP17 transcripts could not be detected in the epididymis after bilateral and unilateral orchiectomy (18). However, mEP17 transcripts were still detected 4 d after unilateral orchiectomy; whereas, in the present study, the protein could not be detected 2 d after castration. This demonstrates that mEP17 present in the fluid at the time of surgery disappeared rapidly either because of its active reabsorption by clear cells and/or its short half-life and/or a translation arrest of the mEP17 transcripts. However, discrepancy in sensitivity between techniques for mRNA and protein detections have to be considered, and further analysis would be needed to characterize the time-course disappearance of both mRNA and protein. In contrast, mE-RABP transcripts are regulated by circulating androgens (16), indicating that two genes that evolved by gene duplication from an ancestral gene display differential regulation of their expression.
Our data indicate that mEP17 gene expression is under the control of luminal testicular factors reaching the initial segment of the epididymis through the efferent ducts. The rete testis fluid contains various soluble molecules (i.e. androgens bound to ABP or Sertoli-derived factors) and spermatozoa; all these components may regulate epididymal gene expression. Indeed, there is evidence that ß-fibroblast growth factor regulates -glutamyl transpeptidase gene expression (42) and that sperm-associated factor(s) regulate proenkephalin gene expression (40). Translocation of the testis in the abdominal cavity, and the subsequent rise in testicular temperature, induces spermatogenic arrest. In this study, the expected spermatogenic arrest was observed, and mEP17 was barely detectable in the epididymis, suggesting that germ cell-associated factor(s) are required for mEP17 gene expression. However, because the epididymis is also translocated in the abdominal cavity, the temperature increase may have a deleterious effect on epididymal gene expression as shown in vitro for the protein CD52 (43). In addition, the secretion of Sertoli cells-derived factors may be affected by the increase in temperature. To dissociate the effect of elevated temperature on the epididymis, the Sertoli cells, and the germ cells, mEP17 expression was determined after a single busulfan injection of 10 mg/kg body weight, known to cause a transient loss of A1 spermatogonia (29). Thirty-five days after injection, the wave of germ cell depletion had reached the pachytene spermatocytes from stage I to stage VIII. Although late germ cells were still observed, mEP17 expression was suppressed. The pachytene spermatocytes in stage VIII, 35-d after injection, would have completed spermiogenesis two seminiferous epithelium cycles (8.6 d) later, i.e. 52.2 d after injection. Indeed, 55 d after busulfan injection, no testicular spermatozoa were released in the epididymal lumen, but there was a resumption of spermatogenesis, and mEP17 gene expression increased. Once again, it suggests not only that impaired spermatogenesis inhibits mEP17 gene expression but that the presence of early germ cells, rather than late germ cells, is important for mEP17 gene expression. However, a toxic effect of busulfan on epididymal cells cannot be completely excluded.
During postnatal development, mE-RABP expression is detected at 3 wk, as blood androgen levels increase (44, 45). Although a faint expression of mEP17 was observed by immunohistochemistry in 3-wk-old mice, expression of mEP17 seems to be delayed, compared with that of mE-RABP, because mEP17 is detected by Western blotting only in 4-wk-old mice, whereas mE-RABP is clearly detected at 3 wk. The onset of mEP17 expression is similar to that observed for another testicular factors-dependent gene (40). This time window corresponds to the entrance of the first spermatozoa in the epididymis (44). Collectively, these results suggest that normal spermatogenesis is required for mEP17 gene expression and that germ cell associated factor(s) may regulate mEP17 gene expression.
Putative function of mEP17
Our study demonstrates that the mEP17 transcripts are translated in vivo. The immunostaining corresponding to mEP17 gradually decreased in the epididymal fluid from segment 2 to segment 3 of the caput epididymidis, whereas mE-RABP accumulation in the luminal fluid increases from segment 2 to segment 5 of the caput epididymidis (32). Whether mEP17 and mE-RABP have the same ligand(s) is still unknown, but the partial overlapping of mEP17 and mE-RABP in the luminal fluid suggests a possible relay function in ligand binding between these proteins and further suggests that their function may not be redundant.
The ligand of mEP17 is unknown at the present time. Because it is closely related to mE-RABP, which has been shown to bind retinoic acid in vitro (35), it may be involved in retinoid trafficking in the epididymis. Other lipophilic molecules, including steroids and fatty acids, are also putative ligands for lipocalins (7, 8). Androgens, as well as estrogens, are present in testicular fluid and luminal fluid of the proximal epididymis (27). Androgens are bound to the ABP that is reabsorbed by the epithelium of the proximal epididymis (46, 47). It has been suggested that the lipocalin PGDS is an androgen carrier in epididymal fluid (24). One may hypothesize that other sex hormone carriers are present in the epididymal fluid, and that mEP17 has such a function.
During sperm epididymal transit, a significant plasma membrane remodeling occurs, corresponding mostly to phospholipid fatty acid saturation state changes, and subsequent phospholipids release (4). Some lipocalins, such as the sperm-associated lipocalin 24p3, can bind fatty acids as well as retinoids (15). Therefore, the mEP17 lipocalin may also function as a fatty acid carrier protein in the epididymal fluid.
Finally, sperm metabolism generates oxygen-reactive species that, in turn, induce cell damage, including DNA breaking and lipid peroxidation. A tear lipocalin was shown to have a scavenger function by sequestrating various lipid peroxidation products (48). Belonging to the lipocalin superfamily, mEP17 may have a similar function. Further studies are required to identify the physiological ligands of mEP17.
In conclusion, our study demonstrates that mEP17 is secreted by the principal cells of the initial segment as a 22-kDa glycoprotein. This protein is likely reabsorbed by clear cells, and the accumulation of this protein into the luminal fluid is restricted to the proximal caput epididymidis. mEP17 expression is dependent on testicular factor(s) produced during normal spermatogenesis. Although mE-RABP and mEP17 genes were generated by tandem in situ duplication (16, 18), these two epididymis-specific lipocalins show different localization and regulation in the epididymis. The duplication and conservation of differently regulated epididymal lipocalins suggest an important and nonredundant function in male fertility.
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Acknowledgments |
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Footnotes |
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Abbreviations: ABP, Androgen-binding protein; Amp, ampicillin; 1-D, one-dimensional; 2-D, two-dimensional; 3-D, three-dimensional; E-RABP, epididymal retinoic acid binding protein; LB, Luria-Bertani; MALDI, matrix-assisted laser desorption ionization; mEP17, mouse epididymal protein of 17 kDa; mE-RABP, mouse epididymal retinoic acid binding protein; MS, mass spectrometry; MS/MS, tandem MS; nanoES, nanoelectrospray; PGDS, prostaglandin D2 synthase; pI, isoelectric point; QqTOF, quadruple-TOF; RmEP17, recombinant mEP17; RmE-RABP, recombinant mE-RABP; TFA, trifluoroacetic acid; TOF, time-of-flight.
Received September 5, 2002.
Accepted for publication November 20, 2002.
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References |
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1. Biol Reprod 56:985–990[Abstract]
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), in the apical border of the epithelial cells. Arrowheads indicate the strongly stained clear cells and apical cells. n, Nucleus. The segments are numbered according to Soranzo et al. (
