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Biology of Reproduction 61, 264-273 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Hamster Sperm Protein, P26h: A Member of the Short-Chain Dehydrogenase/Reductase Superfamily1

Christian Gaudreaulta, Christine Légaréa, Bruno Bérubéa, and Robert Sullivan2,a

a Centre de Recherche en Biologie de la Reproduction and Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Ste-Foy, Quebec, Canada G1V 4G2


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For successful fertilization to occur, mammalian spermatozoa must undergo a series of modifications in order to reach and penetrate the oocyte. Some of these modifications occur during passage through the epididymis, the site where spermatozoa acquire their fertilizing ability. We have previously described hamster sperm protein, P26h, which is acquired by spermatozoa during epididymal transit, and have proposed that this protein is involved in sperm-egg binding. In the present study, we report the cloning and characterization of the full-length cDNA encoding hamster P26h. A database search using the predicted hamster P26h amino acid sequence revealed 85% identity with mouse AP27 protein and porcine carbonyl reductase, members of the short-chain dehydrogenase/reductase (SDR) family of proteins. Northern blot analysis revealed a major P26h 1-kilobase transcript in the testis. No signal was detected in other somatic tissues of the hamster. In situ hybridization experiments revealed that the P26h gene was predominantly transcribed in seminiferous tubules of the testis and at a lower level in the corpus epididymidis. The identity of the cloned P26h was confirmed by immunoprecipitating in vitro-translated P26h using polyclonal antiserum raised against purified hamster sperm P26h. Taken together, these results identify P26h as a new member of the SDR family of proteins involved in the processes of mammalian gamete interactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fertilization is a highly orchestrated process that begins with the activation of an oocyte by a spermatozoon [1]. Although testicular spermatozoa are fully differentiated cells, they are unable to fertilize an oocyte. In order to acquire this property, spermatozoa must undergo posttesticular modifications within the epididymis [2–5]. During this transit, the male gamete is subjected to major surface modifications such as changes in lipid composition [6] and acquisition of new epididymal proteins [7], as well as posttranslational modifications of existing sperm proteins [8]. Together, these modifications are prerequisites for the spermatozoon's acquisition of its fertilizing ability. These processes are regulated by the epididymal luminal microenvironment, which is influenced by both epididymal and testicular protein synthesis and secretions [9, 10].

Using the hamster as a model, we have previously described a 26-kDa protein, termed P26h, that shows immunocontraceptive properties in active immunization of male hamsters [11]. This protein is localized on the sperm acrosome and is acquired during epididymal transit [7]. P26h plays a role in sperm-egg interactions as shown by the ability of anti-P26h IgG and the corresponding Fab fragment to inhibit sperm-zona pellucida binding, in vivo and in vitro [11, 12]. The human homologue of hamster P26h, P34H, has been proposed as a marker of male fertility [13, 14].

In the present study, we have cloned hamster P26h and characterized its expression pattern along the male reproductive tract by Northern blot and in situ hybridization experiments. Sequence analysis suggests that P26h may be a member of the short-chain dehydrogenase/reductase (SDR) family of proteins.


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

Sexually mature golden hamsters (Mesocricetus auratus; Charles River Inc., St. Constant, PQ, Canada) were used in this study. Hamsters were killed by CO2 inhalation, and the epididymides were excised, defatted, and dissected into caput, corpus, and cauda segments. Tissues were frozen in liquid nitrogen and stored at -80°C until used. Testicular and somatic tissues were processed in the same way. For in situ hybridization, fresh tissues were rinsed in diethyl pyrocarbonate (DEPC)-treated PBS and fixed at 4°C for 2 h in 4% (w:v) paraformaldehyde freshly prepared in PBS. Tissues were cryoprotected by sequential incubations in 10% glycerol for 1 h at 4°C under agitation and then overnight in 50% OCT (embedding medium; Sakura Finetek, Torrence, CA). Tissues were embedded in OCT and frozen in liquid nitrogen. Cryosections of ~7 µm were collected on poly-L-lysine-coated slides, air dried at -20°C, and stored at -80°C until further use.

N-Chlorosuccinimide (NCS) Proteolysis

Proteins from cauda epididymal spermatozoa or from the epididymal fat pad were extracted with 0.5% Nonidet P40 (Sigma, Missisauga, ON, Canada) as previously described [12] and subjected to preparative SDS-PAGE according to Laemmli [15]. After Coomassie blue staining, bands corresponding to a molecular mass of 26 kDa were excised, washed twice with H2O, and rinsed with washing solution (50% [w:v] urea, 50% [v:v] ethanol). The polyacrylamide bands were incubated for 30 min in 20 mg/ml NCS in washing solution, rinsed in water, and then incubated three times, for 1 h each, in an equilibrium solution (0.0625 M Tris-HCl, pH 6.8, 20% [v:v] glycerol, 30% [v:v] ß-mercaptoethanol, 6% [w:v] SDS). The band was then loaded on a discontinuous polyacrylamide gel and subjected to electrophoresis [15]. Patterns of protein fragments were visualized by silver nitrate staining [16] or were Western blotted [17] using a P26h antiserum [18]. Western-blotted P26h fragments were also used for N-terminal sequencing as described below.

Partial Amino Acid Sequence Analysis

P26h was purified as previously described [19] and absorbed on a piece of nitrocellulose sheet. One hundred microliters of cyanogen bromide (CNBr; 50 mg/ml in 70% formic acid) was added to 1 mg of the dry protein and incubated under nitrogen in the dark for 24 h [20]. Digested peptides were loaded onto a reversed-phase C18 column (250 x 1 mm) that was preequilibrated with 0.1% (v:v) trifluoroacetic acid (TFA) in water, and proteins were eluted with a 2–100% gradient of 0.08% (v:v) TFA in 80% acetonitrile. Fractions of 0.5 ml or smaller were collected at a flow rate of 50 µl/min. Protein sequence determination was performed on aliquots from one peak by automated Edman degradation using a pulsed-liquid phase sequencer (Applied Biosystems, Foster City, CA; model 473A).

RNA Extraction

Tissues were homogenized with an Ultra-Turrax T25 (Interscience, Markham, ON, Canada) in 1.5 ml of a fresh homogenization buffer solution (4 M guanidium thyocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M ß-mercaptoethanol). One milliliter of cesium chloride homogenization buffer (2 g of CsCl/2.5 ml) was added to the tissue lysates, layered onto a CsCl cushion (5.7 M CsCl, 0.1 M EDTA, pH 7.5), and centrifuged at 35 000 x g overnight. The RNA pellet was resuspended in TES solution (10 mM Tris-HCl, 5 mM EDTA, 1% SDS, pH 7.4) and extracted with phenol:chloroform 1:1 (v:v) and chloroform:alcohol isoamyl 24:1 (v:v). RNA was precipitated with 0.1 volumes of sodium acetate (3 M, pH 5.2) and 2.5 volumes of 95% ethanol. The RNA pellets were resuspended in DEPC-treated water and quantitated at 260 nm. The RNA quality was evaluated by agarose gel electrophoresis.

Northern Blot Analysis

Total RNA (20 µg) prepared from various tissues was subjected to electrophoresis on 1% agarose-formaldehyde gel and then transferred to a nylon membrane (Qiagen, Santa Clarita, CA) using 20-strength saline-sodium citrate (SSC; 3 M NaCl, 0.3 M sodium citrate), air dried, and UV cross-linked. Membranes were prehybridized at 42°C for 4 h in 50% (v:v) formamide, 0.75 M NaCl, 0.05 M NaH2PO4, 0.005 M EDTA, double-strength Denhardt's reagent (0.2% [w:v] Ficoll 400, 0.2% [w:v] polyvinylpyrrolidone, 0.2% [w:v] BSA), 0.2 mg/ml herring sperm DNA (Sigma), 0.1% SDS, and 8% dextran sulfate. The membrane was hybridized overnight at 42°C in the same solution containing [{alpha}-32P]dCTP-labeled DNA probes. Membranes were washed twice in 0.1-strength SSC, 0.1% SDS at room temperature (RT), washed a third time (30 min) at 65°C in 0.1-strength SSC-0.1% SDS, and exposed to X-Omat (Eastman Kodak, Rochester, NY) film. An RNA ladder (Gibco, Burlington, ON, Canada) provided size standards, and a mouse cyclophilin probe was used as loading control [21].

Western Blot

Proteins from cauda epididymal spermatozoa and testis were extracted as described above and subjected to SDS-PAGE [15]. Briefly, proteins from the polyacrylamide gel were transferred on a nitrocellulose sheet (Bio-Rad, Hercules, CA) [17] and blocked with 5% skim milk in PBS for 1 h. After blocking, the membrane was washed for 10 min in PBS+0.01% Tween 20 (ICN, Montréal, PQ, Canada) and incubated with the anti-P26h antiserum diluted 1:1000 in PBS supplemented with 2.5% goat serum (ICN) for 1 h [18]. The membrane was further washed three times in PBS+0.01% Tween 20 for 7 min and incubated in peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:3000 in PBS supplemented with 2.5% goat serum. Finally, the membrane was washed three times in PBS+0.01% Tween 20, and immune complexes were visualized using a chemiluminescent substrate of peroxidase according to the supplier's instructions (ECL; Amersham, Life Science, Oakville, ON, Canada).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Production of a P26h cDNA Probe

The first amino acid sequence obtained (MKLNFSXL-RLVTGAGKGIG) was subjected to BLAST search and showed a homology of 70% with the peptide sequence of AP27 protein, a marker of adipocyte differentiation [22]. From the nucleic acid sequence of AP27, two primers were selected according to OLIGO 4.01 primer analysis software (National Biosciences, Plymouth, MN) and chemically synthesized (sense downstream 5'-GTG ACA GGG GCA GGG AAA GGG-3' and antisense upstream 5'-GCA ACT GAG CAG ACT AGG AGG-3'). These primers were used in RT-PCR with total RNA from hamster testicular tissues. In brief, 5 µg of total testis RNA was incubated with 0.5 µg oligo(dT) primer at 70°C for 10 min in a final volume of 12 µl and then cooled slowly at RT for annealing. To obtain first-strand cDNA samples, 4 µl of first-strand buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2), 2 µl dithiothreitol (0.1 M), 0.5 µl dNTP (20 mM), and 1 µl Superscript reverse transcriptase (Gibco) were added to the annealing mixture for a total volume of 20 µl and incubated for 60 min at 42°C. A partial P26h cDNA was amplified by PCR using the cDNA strand primers described above (RT template). Each amplification reaction contained 5 µl of RT template (or water as negative control), 5 µl of 10-strength buffer (100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl), 0.2 mM dNTPs, 10 µM of each primer, and 1.5 U Taq polymerase (Pharmacia Biotech, Baie D'Urfé, PQ, Canada) in a final volume of 50 µl. The PCR cycling conditions chosen were 1 min at 95°C, 1 min at 60°C, 1 min at 72°C for 30 cycles, followed by a 5-min extension period at 72°C. The reaction products were analyzed on 1% agarose gel electrophoresis, and the bands were visualized by ethidium bromide staining.

The PCR product (~715 base pairs [bp]) was purified (Quiaquick; Qiagen), cloned in PCR 3.5 plasmid (Invitrogen, San Diego, CA), and digested with EcoRI. The insert (715 bp) was separated from the vector by agarose gel electrophoresis, isolated from the gel with Na45 membrane (Schleicher & Schuell, Inc., Keene, NH), and finally labeled with 32P by random priming using the T7 Quick-Prime kit (Pharmacia Biotech).

Cloning and Sequencing of the P26h cDNA

Poly(A)+ RNA from testis RNA was purified using a PolyAtract RNA purification kit (Promega, Madison, WI) according to the supplier's instructions. A testis cDNA library was prepared according to the instructions provided by the supplier. In brief, testicular poly(A)+ RNA was reverse transcribed and ligated into the lambda Uni-Zap XR vector (Stratagene, La Jolla, CA). The lambda library was packaged using Gigapak III (Stratagene), amplified using Escherichia coli XL1-blue cells, and screened with the random prime-labeled 715-bp P26h cDNA fragment. Positive clones were isolated by plaque purification, and the longest one (1081 bp) was subcloned into pBluscript KS+. All nucleotide sequences were determined by the dideoxinucleotide termination method using a T7 Sequenase version 2.0 kit (Amersham). Search for homologies was performed using BLAST search software.

In Situ Hybridization

Tissue cryosections were fixed with freshly prepared 4% paraformaldehyde in PBS for 5 min at RT, incubated for 10 min in 95% ethanol/5% acetic acid at -20°C, and rehydrated by successive baths in decreasing concentrations of ethanol diluted in DEPC-treated H2O. Target RNA was unmasked by enzymatic digestion with 10 µg/ml proteinase K (Boehringer Mannheim, Laval, PQ, Canada) in PBS for 10 min at 37°C, followed by a 5-min incubation in 0.2% glycine. Sections were postfixed for 5 min with 4% paraformaldehyde in PBS; they were then acetylated with 0.25% acetic anhydride, 0.1 M triethanolamine, pH 8.0, for 10 min, and finally washed with PBS.

Tissues were prehybridized for 1 h with a preheated hybridization solution (0.3 M NaCl, 0.01 M Tris-HCl, pH 7.5, 1 mM EDTA, single-strength Denhardt's solution, 5% dextran sulfate, 0.02% SDS, and 50% formamide) containing 250 µg/ml salmon sperm DNA. Sections were then incubated overnight at 42°C with 25 µl (5 µg/ml) of heat-denatured antisense or sense digoxigenin (DIG; Boehringer) cRNA probe according to the supplier's instructions. After incubation, sections were washed twice in double-strength SSC (0.3 M NaCl, 0.03 M trisodium citrate) at RT, followed by two 10-min washes at 42°C in double-strength SSC, single-strength SSC, and 0.2-strength SSC. Hybridization reactions were detected by immunostaining with an alkaline phosphatase-conjugated anti-DIG antibody. Nonspecific staining was blocked by incubation for 1 h with 5% (v:v) heat-inactivated sheep serum in 0.2 M Tris-HCl, 0.2 M NaCl, and 0.3% Triton X-100. Sections were subsequently incubated for 2 h at RT with the alkaline phosphatase-conjugated anti-DIG antibodies diluted 1:1000 in blocking solution; washed with 0.2 M Tris-HCl, 0.2 M NaCl buffer; and finally incubated with 0.1 M Tris-HCl, 0.1 M NaCl, and 0.01 M MgCl2 at pH 9.5. The hybridization signal was visualized after a 10- to 15-min incubation with the substrates nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Gibco). Levamisole (2 mM; Sigma) was added to the reaction mixture to inhibit endogenous alkaline phosphatase. Tissue sections were immersed in 1 mM EDTA, 0.01 Tris-HCl, pH 7.5; they were then washed for 5 min in H2O, counterstained with neutral red, dehydrated through graded alcohols, cleared in xylene, and mounted with Permount (Fisher Scientific, Nepean, ON, Canada).

Eukaryotic In Vitro Translation

In vitro transcription/translation was performed using a plasmid containing the full-length P26h cDNA. The TNT-coupled reticulocyte lysate system was used according to the supplier's instructions (Promega). In brief, 0.5 µg of circular plasmid DNA was added directly to TNT rabbit reticulocyte lysate. T3 RNA polymerase (Promega) and [35S]methionine (10 mCi/ml) were added to the transcription/translation mixture. The reaction was allowed to proceed for 2 h at 30°C. Newly synthesized proteins were analyzed by SDS-PAGE. The gel was soaked in an enhancer solution (Amersham), dried, and then exposed to X-Omat AR film (Kodak) for 6 h at RT. In some experiments, the translation products were subjected to NCS proteolysis (as described above) before electrophoretic analysis. In other experiments, the translation products were immunoprecipitated using a P26h antiserum. Fifteen microliters of the translation reaction mixtures was incubated for 1 h at RT with the P26h antiserum [11] or a control serum, both diluted (1:13) in Tris-saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Fifty microliters of packed protein-A sepharose (Pharmacia) was then added for a total volume of 1 ml, and the mixtures were incubated for an additional hour at RT. Immunoprecipitates were washed several times in Tris-saline (50 mM Tris-HCl, 500 mM NaCl, pH 7.5). Immune complexes were dissociated in SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.3, 2% SDS [w:v], and 5% [v:v] ß-mercaptoethanol) and subjected to SDS-PAGE. The gel was enhanced, dried, and exposed to X-Omat AR film (Kodak) for 12 h at RT.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
When purified P26h was subjected to Edman degradation, 27 of the 29 amino acids generated were identified in the N-terminal. A 17-kDa fragment obtained by NCS proteolysis of P26h allowed identification of 15 of the 26 amino acids analyzed, whereas the fragment obtained following CNBr treatment allowed the identification of 8 of 9 additional amino acids. From a total of 40 amino acids identified by Edman degradation of P26h peptides, 37 showed homology with a mouse AP27 sequence (Fig. 1). This protein has been shown to be a differentiation growth factor of mouse adipoblasts [22].



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  		FIG. 1. A) Partial amino acid sequences of P26h. Sequences were obtained by Edman degradation from purified P26h-, NCS-, and CNBr-generated fragments. B) Partial sequences were compared with the mouse AP27 amino acid sequence deduced from its cDNA. Identical amino acids are marked with large dots; gaps are marked with hyphens (-), and undetermined amino acids are marked with X.

A 26-kDa band from SDS-PAGE analysis of the fat pad protein extract was digested with NCS as described for the P26h protein. Unlike P26h, the NCS 26-kDa protein obtained from the fat pad did not yield a digestion pattern similar to the one obtained with the P26h. Moreover, only the P26h and its NCS-digested fragment were detected by a P26h antiserum in Western blot analysis (Fig. 2). An NCS-digested fragment of P26h was sequenced and showed high homology to mouse AP27 (Fig. 1). The intact 26-kDa protein from the fat pad protein extract was subjected to the same procedure, and no N-terminal sequence was obtained.



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  		FIG. 2. SDS-PAGE and Western blot analysis of intact (lanes 1–3) and NCS-treated proteins (lanes 4–6). A) Silver-stained SDS-PAGE pattern of intact albumin control (lane 1), 26-kDa band of the epididymal fat pad (lane 2), P26h from cauda epididymal spermatozoa (lane 3), and peptides generated by NCS proteolysis of albumin (lane 4), 26-kDa band of the epididymal fat pad (lane 5), and P26h from cauda epididymal spermatozoa (lane 6). B) The corresponding Western blot probed with the anti-P26h antiserum.

Oligonucleotides derived from the cDNA sequence of mouse AP27 were used to amplify a 715-bp P26h cDNA fragment as described in Materials and Methods. This 715-bp fragment was subsequently cloned and sequenced. Search for homologues using BLAST revealed 85% homology with the AP27 cDNA (not shown). The tissue distribution of the P26h transcript gene was determined using this fragment as a probe in Northern analysis. Using total RNA obtained from several tissues, a 1081-bp message was detected solely in the testis (Fig. 3A). Again no transcript was found in the fat, confirming that the P26h cDNA did not originate from this tissue. To confirm the amount and integrity of the RNA in all samples, the same blot was reprobed with a ubiquitously expressed random cyclophilin cDNA probe. Western blot using P26h antiserum performed on extracted proteins from cauda epididymal sperm and testicular tissues showed a 26-kDa band in both extracts (Fig. 3B).



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  		FIG. 3. A) Northern blot analysis of total hamster RNA from 1) testis, 2) whole epididymis, 3) caput epididymides, 4) corpus epididymides, 5) cauda epididymides, 6) fat pad, 7) lung, 8) heart, 9) liver, 10) kidney, 11) muscle, and 12) brain probed with a P26h 715-bp cDNA probe (top) or with a cyclophilin probe as a positive control (bottom). B) Western blots, using P26h antiserum, performed on proteins extracted from testicular tissues (lane 1) and cauda epididymal spermatozoa (lane 2). Cauda epididymal sperm proteins were used as positive control. Molecular weight markers (x 10-3) are on the left.

To obtain a full-length cDNA clone, a cDNA library was constructed in lambda gt11 from hamster testicular mRNA. A total of 1 x 105 clones were screened with the 715-bp cDNA probe. Of 32 primary positive clones, 11 were further characterized by PCR. The longest insert was cloned into a Bluescript SK phagemid and sequenced. The 1081-bp P26h cDNA has a 732-bp open reading frame, starting with an ATG at position 124, and a stop codon at position 856, followed by a polyadenylation signal and poly(A) tail (Fig. 4). The deduced amino acid sequence predicted a 26-kDa protein that was in agreement with the molecular mass of the P26h purified protein as determined by SDS-PAGE. The N-terminal sequence of P26h and peptides generated by Edman degradation (Fig. 1) were also in agreement with the P26h predicted amino acid sequence (Fig. 5). The P26h amino acid sequence was compared with the AP27 and a carbonyl reductase, which showed homology of 87% and 80%, respectively. AP27 and carbonyl reductase are members of the SDR family of proteins. P26h also showed conserved patterns characteristic of SDR proteins, i.e., the NADH or NADPH (b-nicotinamide adenine dinucleotide phosphate) coenzyme-binding site and the active site, which are GlyXXXGlyXGly and TyrXXXLys, respectively (Fig. 5).



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  		FIG. 4. Nucleotide sequence of the P26h cDNA cloned from testicular cDNA of a mature hamster. The nucleotides are numbered from the 5' end of the cDNA. The translation of the proposed open reading frame is shown below the nucleotide sequence and encoded a 244-amino acid protein ending with an amber codon. Underlined is the polyadenylation signal.



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  		FIG. 5. Amino acid sequence comparison of hamster P26h with mouse AP27 and pig carbonyl reductase. The P26h protein sequence was deduced from its cDNA and compared with the deduced amino acid sequences of the AP27 and carbonyl reductase. Only different amino acids are shown; identical amino acids are marked with hyphens (-).

In situ hybridization was used to identify which testicular compartment and cell type express the P26h gene. P26h mRNA was found in the spermatogenic cells in seminiferous tubules of testis from sexually mature hamsters. Using nonradioactive immunostaining, in addition to the testis, in situ hybridization also revealed a weaker signal along the epididymis, principally in the corpus region (Fig. 6).



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  		FIG. 6. In situ hybridization localization of the P26h mRNA along the male reproductive tract, probed with the antisense P26h RNA probe. A) Histological sections of hamster testicular tissues. B) High magnification of testicular tissues, arrows showing stained spermatogenic cells; I, interstitial compartment of testicular tissues; and L, the seminiferous tubule lumen. C) Caput epididymidis. D) Corpus epididymidis. E) Cauda epididymidis. F) Testicular tissues probed with the sense P26h RNA probe used as a negative control. P26h mRNA was detected with the DIG anti-DIG complexes resulting in blue staining.

The cloned P26h was used to translate the protein using an in vitro transcription/translation system. The P26h plasmid produced translation products of 26 kDa on SDS-PAGE (Fig. 7A, lane 3). Total translation products were also subjected to immunoprecipitation using a P26h antiserum. The P26h antiserum recognized a single 26-kDa protein on SDS-PAGE (Fig. 7A, lane 2), suggesting that the cloned P26h corresponded to the actual P26h protein. No signal was detected on control performed with preimmune antiserum. Total translation products were equally subjected to NCS proteolysis. NCS proteolysis generated a 17-kDa fragment on SDS-PAGE in agreement with the deduced amino acid sequence and the NCS proteolysis of purified P26h (as shown in Fig. 2).



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  		FIG. 7. Immunoprecipitation of P26h cDNA translation products. A) SDS-PAGE of P26h cDNA translation products immunoprecipitated with a preimmune (lane 1) or the P26h antiserum (lane 2). Lane 3 shows total translation products. B) The 26-kDa translational products were reanalyzed by SDS-PAGE before (lane 1) or after NCS proteolysis (lane 2). Proteins were labeled with [S35]methionine and detected by fluorography. Molecular weight markers (x 10-3) are on the left.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During epididymal transit, mammalian spermatozoa acquire their fertilizing ability. One of the best-documented physiological functions acquired by spermatozoa during epididymal maturation is their ability to efficiently interact with the egg's zona pellucida [23–25]. Our laboratory has been interested in sperm surface modifications, i.e., the addition of new surface proteins, or the posttranslational modification of preexisting sperm components, that are necessary to produce a functional male gamete. Using the hamster as a model, we have previously identified a sperm protein, P26h, that shows affinity for homologous zona pellucida glycoproteins [12]. P26h is abundant in the luminal fluid of the proximal region of the hamster epididymis, its concentration decreasing along the length of the tubule [7]. In parallel, P26h accumulates on spermatozoa during epididymal maturation [26]. P26h is phosphatidylinositol anchored to the sperm plasma membrane during epididymal transit, and prostasome-like particles may be involved in this transfer [27]. P26h is exclusively located on the surface covering the acrosomal cap of the mature spermatozoa, the subcellular domain involved in zona pellucida binding [11].

In this study, P26h was purified after detergent extraction of cauda epididymal spermatozoa. This was performed by preparative SDS-PAGE [11] as well as by chromatographic procedures [19]. In the latter case, a single spot following two-dimensional gel electrophoresis was obtained; this single protein is recognized by an anti-P26h serum on corresponding Western blots [19]. These two independent preparations of purified P26h, as well as proteolytic fragments, were N-terminal sequenced by Edman degradation. All amino acid sequences obtained showed high homology with mouse AP27 (Fig. 1). AP27 has been described as a differentiation factor of adipoblasts in adipocytes [22]. AP27 mRNA has been shown to be expressed by TA1 and 3T3-L1, two adipogenic stem cell lines potentially able to differentiate into adipocytes [28, 29]. In the hamster, as well as in the mouse, the majority of the epididymis is surrounded by a fat cushion [30]. Considering that the purified P26h was obtained from spermatozoa recovered from the distal cauda epididymidis, we were concerned about the possibility that the N-terminal sequences were obtained from AP27 liberated from adipocytes contaminating the sperm suspensions. This was conceivable considering that the molecular mass of AP27 as deduced from its mRNA sequence is approximately 27 kDa [22]. Proteins from the epididymal fat pad were extracted and processed in parallel with cauda epididymal spermatozoa. NCS digests were Western blotted and probed with the anti-P26h serum. The 26–27-kDa fat pad protein, as well as the proteolytic fragments generated by NCS, was undetectable by the anti-P26h serum (Fig. 2). Furthermore, no signal was detectable when the 26–27-kDa fat pad protein was N-terminal sequenced by Edman degradation (data not shown). From these results we can conclude that the N-terminal sequences obtained did not result from contamination of the sperm preparation by the epididymal fat pad.

Northern blot analysis revealed a major P26h transcript in testicular tissues of the sexually mature hamster. This mRNA is undetectable in the other tissues analyzed, including the fat pad and the epididymis (Fig. 3A). Western blots, using P26h antiserum, showed a 26-kDa band in the testicular protein extract, confirming that P26h mRNA is translated in the testis (Fig. 3B). It has been previously demonstrated that P26h protein is absent in various somatic tissues of the hamster [11]. The fact that P26h mRNA was undetectable on Northern blots of epididymal RNA was unexpected, since Robitaille et al. [7] previously reported that an in vitro translation product encoded by mRNA of the proximal region of the hamster epididymis can be immunoprecipitated by anti-P26h antibodies. In this study, in situ hybridization confirmed that the P26h transcript is expressed in the testis by spermatogenic cells and, at a lower level, in the epididymis. In situ hybridization was performed by means of a DIG-labeled RNA probe system using an anti-DIG antibody that allows amplification of the signal and thus provides more sensitive mRNA detection than the traditional Northern blot analysis. Faint labeling is detectable all along the epididymis, with a much stronger signal being associated with the corpus (Fig. 6). In many species, the proximal corpus region is known to be the more active epididymal segment for protein synthesis and secretion [31–33]. According to the Northern blot analysis (Fig. 3), P26h, which is found at high concentrations in the proximal region of the hamster epididymis [7], probably originates from testicular fluid as a secretory product of the seminiferous tubules, as suggested by the in situ localization of the transcript (Fig. 6). This protein may also be secreted by the corpus epididymidis. A dual testicular and epididymal origin has been described for other proteins interacting with the spermatozoa during epididymal maturation [34–36]. Whether or not the testicular and the epididymal P26h are identical or exist as different isoforms, as has been described for clusterin [37, 38], remains to be determined.

Since P26h was expressed principally in testicular tissues (Fig. 3), a testicular cDNA was screened to clone the full-length P26h cDNA. The longest transcript obtained from the library was sequenced; it revealed a cDNA of 1081 bp coding for a 244-amino acid protein. The predicted size of the translational product was in agreement with the electrophoretic behavior of P26h extracted from cauda epididymal spermatozoa. The P26h cDNA showed high sequence homology with AP27, as expected from N-terminal amino acid sequences, and with a carbonyl reductase (Fig. 5) known to be expressed in the pig lung [39] (Fig. 5). The sequence homology between P26h cDNA and the cDNA encoding for these two proteins is 86% and 85%, respectively. The deduced amino acid sequence also shows a high homology of 87% with the AP27 and 80% with the carbonyl reductase. Considering the suggestion that P26h is involved in sperm-zona interactions [11, 12], the biological function attributed to these two proteins to date was puzzling. AP27 has been described as a differentiation factor of adipoblasts, and its expression has been shown to be inhibited by activators of protein kinase C [22]. The carbonyl reductase is a homotetramer that catalyzes the oxidation of secondary alcohols and aldehydes [40–46]. This enzyme has been shown to be expressed specifically in the lung and mainly distributed in the mitochondria [39]. Despite the high level of homology with AP27 and carbonyl reductase, P26h showed a completely different tissue distribution. P26h protein and its encoding mRNA are not expressed in the lung or in the adipocyte (Figs. 2 and 3; [11]). AP27 and carbonyl reductase are known to be members of the SDR superfamily. Interestingly, P26h shows some of these properties [47].

The SDR superfamily is formed by a variety of proteins that exhibit residue identities of only 15–30%. This low level of sequence identity between the members indicates an early divergence. This is reflected by the wide range of functions fulfilled by the members of this superfamily. There are three classes of enzymes covering a wide range of EC numbers, i.e., 1, 4.2, 5.1, and 5.3, as well as members with unknown functions [47–49]. Two consensus sequences are conserved in this family, the NAD(H) or NADP(H) binding domain, an N-terminal segment (GlyXXXGLYXXGly), and the catalytic domain (TyrXXXLys) [47]. Interestingly, the P26h deduced amino acid sequence possesses these consensus domains as well as the Gly 129, Ser 136, and Pro 179, which are conserved in more than 90% of the SDR family members [50] (Fig. 5).

Polyclonal antibodies have been produced against P26h and used to document the function of this sperm protein during the fertilization process in the hamster. When added to an in vitro fertilization medium, anti-P26h antibodies inhibit the sperm-zona pellucida interaction in a dose-dependent manner [11, 51]. Furthermore, active immunization of male hamsters against the purified P26h results in an immune response associated with reversible infertility [11]. Using the P26h antiserum, a human counterpart of P26h has also been identified [52] and shown to be absent from sperm of men with idiopathic infertility [18]. In humans, this protein is also acquired by the spermatozoa during epididymal transit [14, 52]. Taken together, these results clearly demonstrate the involvement of this sperm protein in the processes leading to fertilization. The P26h preparation that shows immunocontraceptive properties was the same as that used to determine N-terminal sequence by Edman degradation (Fig. 1). Furthermore, the polyclonal antiserum that allowed us to document the function of P26h in the process of fertilization also reacts with the translation product encoded by the sequenced P26h cDNA (Fig. 7). This clearly demonstrates that this SDR member is involved in mammalian sperm-egg interaction.

The mammalian spermatozoon is a highly polarized cell characterized by well-defined membrane domains [1, 53, 54]. Many sperm surface proteins have been proposed as playing a role during the cascade of events occurring when the male gamete reaches the oocyte [55–57]. Various sperm proteins have been proposed as candidates for zona pellucida binding [58, 59], some of which show enzymatic activity, such as proacrosin [60], a trypsin-like protease [61], mannosidase [62], galactosyltransferase [63, 64], and P95 [65], which is a hexokinase [66–68]. The catalytic activity of these enzymes may not necessarily be involved in interaction with the zona pellucida. Rather, it is the substrate affinity that mediates this interaction [69]. The biological function played by these proteins in gamete interactions is thus quite different from their enzymatic activity as defined by their catalytic activity in cell metabolism. This discrepancy is reflected by their subcellular localization on the spermatozoon. To mediate zona pellucida recognition, these enzymes must be localized at the sperm surface, whereas classically they are known to be intracellular. This is well illustrated by the extracellular-oriented sperm membrane mannosidase [70, 71] and galactosyltransferase [72], as well as by hexokinase, which is at the surface of the mouse spermatozoa [73] but usually known in association with the mitochondrial membrane [74]. Like these potential zona pellucida ligands, P26h is localized at the hamster sperm surface, on the membrane domain covering the acrosome [1, 27].

P26h belongs to the SDR superfamily characterized by highly different members with a low level of identity [47, 75]. This reflects distant duplications and early divergence. As a consequence, the SDR family demonstrates great diversity in enzymatic activities and functions [47]. An interesting example of an alternative function for an enzyme is glyceraldehyde-3-phosphate dehydrogenase [76]. This protein, which is classically known as a glycolytic enzyme, has been shown to act as a tRNA-binding protein with a function in cytoplasmic trafficking [77]. SDR divergence is favorable for the emergence of new biological functions [78]; involvement in gamete interactions may be one of these. Considering that P26h has been previously shown to be involved in gamete interaction and to possess immunocontraceptive properties, the relationship between structure and function of this new member of the SDR family may contribute to a better understanding of mammalian fertilization.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. S. St-Jacques for advice regarding in situ hybridization, Mrs. L. Coutu for technical assistance, Dr. Sylvie Bourassa from "Service de séquence de peptides l'est du Québec" (Ste-Foy, PQ) for her expertise in N-terminal sequencing, and Drs. Robert Viger and Janice Bailey for valuable comments and criticisms of the manuscript.


   FOOTNOTES
 
1 This study was supported by the Medical Research Council of Canada grant to R.S. C.G. receives a scholarship from the Medical Research Council of Canada. Back

2 Correspondence: Robert Sullivan, Unité d'Ontogénie-Reproduction, Centre de Recherche, Centre Hospitalier de l'Université Laval, 2705 Blvd. Laurier, Ste-Foy, QC, Canada, G1V 4G2. FAX: 418 654 2765; robert.sullivan{at}crchul.ulaval.ca Back

Accepted: February 26, 1999.

Received: November 16, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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