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Regular Article |
a Centre de Recherche en Biologie de la Reproduction and Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada G1V 4G2
ABSTRACT
P26h is a hamster sperm protein of 26 kDa that has been previously characterized as a surface protein covering the acrosome acquired during epididymal transit. P26h is involved in sperm-egg interactions. Recently, it has been shown that the P26h transcript is highly expressed in the testis, and the P26h cDNA has been cloned from a hamster testicular cDNA library. Herein we report the production of a fusion protein (maltose binding protein-P26h) with the whole P26h cDNA encoding sequence and the production of a polyclonal antiserum against it. In Western blots, this antiserum recognized both the P26h extracted from cauda epididymal spermatozoa and the MBP-P26h. We also determined the age of appearance of P26h and which germ cell types express P26h mRNA and its translational product. Northern blots and in situ hybridization analysis showed that P26h transcripts appear at 3 wk of age, within the first round of spermatogenesis in the golden hamster. In situ hybridization showed that P26h transcripts are expressed in spermatocytes and round spermatids, whereas immunostaining revealed the presence of P26h in the cytoplasm of round spermatids and elongated spermatids. P26h was undetectable in testicular spermatozoa. Both in situ hybridization and immunostaining showed P26h expression to be dependent of the testicular cell type and the epithelium cycle. The implications for P26h in sperm-egg interaction and the testicular origin of P26h are discussed.
epididymis, gametogenesis, sperm, spermatid, testis
INTRODUCTION
Using the hamster as a model, we have previously described a 26-kDa protein, called P26h. This protein is localized on the sperm surface covering the acrosome cap and is acquired by spermatozoa during epididymal transit [1]. P26h is involved in the sperm-egg interaction, as shown by its immunocontraceptive properties when used in active male immunizations and by the ability of both anti-P26h IgGs and corresponding Fab fragments to block sperm-zona pellucida binding [2]. Moreover, similar proteins exist in humans and monkeys, P34H and P31m, respectively, that are both localized on the surface of the acrosome. P34H shares antigenic epitopes with P26h [3], and its deduced amino acid sequence is 65% homologous with the P26h amino acid sequence [4]. Low levels of P34H are associated with male infertility [5]. Recently, we showed that the P26h transcript is strongly expressed in sexually mature testis and at weaker levels in the epididymis [6].
Fully differentiated and fertile spermatozoa are the result of a series of events in the male reproductive tract. First, the production of sperm occurs within the testicular seminiferous tubules, during spermatogenesis. During this process, diploid germ cells, spermatogonia, will mature to become highly differentiated haploid cells, spermatozoa, following a series of biochemical and morphological events [7]. The seminiferous epithelium includes both somatic cells, the Sertoli cells, and germ cells. The Sertoli cells, upon regulation by FSH, participate actively in the maturation of the spermatozoa [8]. In mammals, specific cellular associations are observed throughout spermatogenesis, and these associations can be separated in different stages. In golden hamsters, the cellular associations of the seminal epithelium include 13 different stages [9]. Usually spermatogenesis is separated in three phases, spermatogonial proliferation and renewal, mitosis and meiosis, and spermiogenesis [10]. Especially during spermiogenesis, spermatids undergo drastic modifications such as acrosomal development [11], elongation, and nuclear condensation [12]. Nuclear condensation involves transcription cessation and mRNA storage in order to complete the process of spermiogenesis [13]. Moreover, the translation of stored mRNAs can be regulated as a function of the stage of the epithelium cycle [14]. Following spermatogenesis, spermatozoa will travel to the epididymis, where they will achieve maturation. During this period, spermatozoa will be subjected to major surface modifications leading to the acquisition of fertilizability [15, 16].
In this study, we have undertaken to determine at which age P26h transcripts appear in the testis by performing Northern blot analysis and in situ hybridization, and to analyze which germ cells express P26h mRNA and P26h protein by in situ hybridization and immunostaining, respectively. Then we relate the differential localization of P26h in spermatids and epididymal spermatozoa. The results are discussed with regard to the possible dual functions of P26h during spermatogenesis and its involvement in fertilization.
MATERIALS AND METHODS
Fusion Protein (MBP-P26h)
Fusioned maltose binding protein-P26h (MBP-P26h) was produced using a pMal Protein and Purification System (New England Biolabs [NEB], Missisauga, ON, Canada) according to the supplier's protocol. In brief, the whole P26h cDNA coding sequence [6] was amplified by polymerase chain reaction using specifically engineered oligonucleotides deduced from the encoding P26h cDNA sequence. The upstream oligonucleotide started at the ATG and was flanked with an EcoRI restriction site, and the downstream oligonucleotide terminated at the stop codon and was flanked with a HindIII restriction site. Following amplification, the amplicon was digested with EcoRI/HindIII and cloned into the Pmal-c vector (NEB) digested in the same way. This construction was further incorporated into Escherichia coli DH5
(Gibco BRL, Burlington, ON, Canada). Fusion protein synthesis was induced by the addition of isopropyl-ß-D-thiogalactopyranoside (ICN, Montréal, PQ, Canada) at a concentration of 0.3 mM in culture medium. Induced bacteria were lysed by sonication, and the fusion protein was purified using an affinity chromatography column of amylose resin in accordance with supplier's instructions (NEB).
Antibodies
P26h antiserum was raised in the rabbit against P26h purified from proteins extracted from cauda epididymal hamster spermatozoa as previously described [17]. MBP-P26h antiserum was raised against purified MBP-P26h produced as described below. Rabbits were immunized against 500 µg MBP-P26h in 500 µl of Dulbecco PBS (D-PBS), pH 7.4 (Gibco BRL) emulsified with an equal volume of complete Freund adjuvant (Gibco BRL). Booster injections were administered with 250 µg of MBP-P26h in 500 µl of D-PBS emulsified with an equal volume of incomplete Freund adjuvant (Gibco BRL). Subcutaneous booster injections were repeated monthly until high anti-P26h-IgG titers were reached. Blood was collected by cardiac puncture and clotted at 4°C overnight; serum was recovered by centrifugation and frozen at -80°C until used.
Western Blotting
MBP-P26h and Nonidet P40 (Sigma, Oakville, ON, Canada)-extracted proteins from cauda epididymal spermatozoa [18] were subjected to SDS-PAGE [19], stained with Coomassie blue, or transferred on nitrocellulose membrane (Bio-Rad, Hercules, CA) [20]. Briefly, the nitrocellulose membranes were blocked with PBS, pH 7.4 containing 5% skim milk for 1 h at room temperature (RT). After blocking, the membranes were washed for 10 min with PBS-0.1% Tween 20 (ICN) and incubated with either anti-P26h antiserum diluted 1:1000 in PBS supplemented with 2.5% goat serum (ICN) or with anti-MBP-P26h antiserum diluted 1:5000 in PBS supplemented with 2.5% goat serum (ICN). The membranes were further washed three times in PBS-0.1% Tween 20 for 7 min and incubated with peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:3000 in PBS supplemented with 2.5% goat serum. Finally, the membranes were visualized using a chemiluminescent substrate of peroxidase according to the supplier's instructions (ECL; Amersham, Life Science, Oakville, ON, Canada).
Immunostaining
Spermatozoa were collected from distal cauda epididymidis of sexually mature hamsters (Mesocricetus auratus, Charles River Inc., St-Constant, PQ, Canada), smeared on microscope slides, and air dried. Previous studies showed that P26h is only on the sperm surface [2]. Nonspecific staining was avoided by preincubating the smears with 10% goat serum in PBS. After three washes with PBS, the slides were incubated with the rabbit anti-MBP-P26h recombinant protein or with preimmune serum, both diluted 1:50 in PBS. Spermatozoa were washed three times and incubated for 30 min with a biotinylated goat anti-rabbit IgG (Dimension Laboratories, Mississauga, ON, Canada) diluted 1:100 with PBS. After three washes, smears were incubated with an avidin-biotin peroxidase complex (Vector Labs, Burlingame, CA), incubated for 10 min with 3-amino-9-acetylcarbazole in 50 mM acetate buffer, pH 5.2 in the presence of 0.002% H2O2, and finally, extensively washed in H2O. In order to perform histological sections of testicular tissues, hamsters were anesthetized with ketamine xylazine, and testes were fixed via cardiac perfusion of 0.9% saline supplemented with heparin followed by buffered 10% formaldehyde. Paraffin sections were cleared by three immersions in xylene for 5 min, two in ethanol (100%) for 5 min, and two in ethanol (95%) for 3 min. To neutralize endogenous peroxidase, sections were immersed in methanol (100%) supplemented with hydrogen peroxide (Sigma) at a final concentration of 3% for 30 min. Sections were further immersed for 5 min in ethanol (95%) and in distilled water. In order to block unspecific antibody binding sites, sections were treated with 10% goat serum (ICN) in PBS for 15 min. After blocking, sections were rinsed three times in PBS for 2 min and incubated with either the anti-P26h antiserum or the anti-MBP-P26h, antiserum both diluted 1:100 in PBS for 1 h. Sections were then rinsed three times in PBS and incubated for 30 min with biotinyled goat anti-rabbit immunoglobulins (Dako, Mississauga, ON, Canada) diluted 1:100 in PBS. Finally, sections were rinsed three times with PBS and processed for staining using an ABC vectastain kit (Vector Laboratories, Burlingame, CA) according to the supplier's protocol. Tissues sections were counterstained with hematoxylin (Sigma).
RNA Extraction
Tissues were homogenized with a Polytron in 1.5 ml of a freshly prepared homogenization buffer solution (4 M guanidium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-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 pellets were resuspended in TES solution (10 mM Tris-HCl, 5 mM EDTA, 1% SDS, pH 7.4) and extracted with phenol/chloroform 1:1 (vol/vol) and chloroform/isoamyl alcohol 24:1 (vol/vol). RNA was precipitated with a 0.1 vol of sodium acetate (3 M, pH 5.2) and 2.5 vol of 95% ethanol. The RNA pellets were resuspended in diethyl pyrocarbonate (DEPC)-treated water and quantitated at 260 nm. The RNA quality was evaluated by agarose gel electrophoresis.
Northern Blot Analysis
Twenty-microgram samples of total RNA prepared from different tissues were subjected to electrophoresis on a 1% agarose-formaldehyde gel and then transferred to a nylon membrane (Qiagen, Santa Clarita, CA) using 20x SSC (3 M NaCl, 0.3 M Na-citrate), air dried, and UV cross-linked. Membrane was prehybridized at 42°C for 4 h in 50% (vol/vol) formamide, 0.75 M NaCl, 0.05 M NaH2PO4, 0.005 M EDTA, 2x Denhardt reagent (0.2% [wt/vol] Ficoll 400, 0.2% [wt/vol] polyvinylpyrrolidone, 0.2% [wt/vol] 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 [-32P]dCTP-labeled P26h cDNA probes (715 base pairs [bp]) [6]. Membranes were washed twice in 0.1x SSC, 0.1% SDS at RT (room temperature) followed by a third wash of 30 min at 65°C in 0.1x SSC-0.1% SDS, and exposed to Kodak X-Omat film. An RNA ladder (Gibco) was used as size standards and a bovine actin probe was used as a loading control.
In Situ Hybridization
3H-UTP-labeled probe Thick paraffin sections (30 µm) of the hamster testis were cut, and the unmounted sections were collected in vials filled with toluene for deparaffinization. The floating sections were subsequently rehydrated; postfixed in 2% glutaraldehyde, 4% formaldehyde, and 3% dextran in 0.05 M phosphate buffer; and washed in the same buffer containing 7.5% glycine. The hybridization of the floating sections were performed overnight at 40°C with a 3H-UTP P26h riboprobe. After hybridization, the thick sections were postfixed in osmium tetroxide, flat-embedded in Epon, cut and incubated with liquid photographic emulsion (Kodak NTB-2), and processed after 2 mo of exposure.
Digoxigenin-labeled cRNA probe Testes tissues were obtained from golden hamsters of different ages and processed for cryosectioning [6]. 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 of decreasing concentrations of ethanol diluted in DEPC-treated H2O. Target RNA was unmasked by enzymatic digestion with 10 µg/ml proteinase K (Roche Diagnostics, Laval, QC, 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, 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, 1x Denhardt 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; Roche Diagnostics) cRNA probes, transcribed from the P26h cDNA sequence [6] according to the supplier's instructions. After incubation, sections were washed twice in 2x SSC (0.3 M NaCl, 0.03 M Na3 citrate) at RT, followed by two 10-min washes at 42°C in 2x SSC, 1x SSC, and 0.2x SSC, respectively. 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% (vol/vol) 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 nitroblue 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 M Tris-HCl, pH 7.5, 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).
RESULTS
Two different antibodies raised against the P26h were used in this study: one against the purified P26h and one against the MBP-P26h. When used on Western blots of extracted cauda epididymal sperm proteins, both the anti-P26h and the anti-MBP-P26h serum, recognized a single band of 26 kDa. Moreover, both antibodies recognized the MBP-P26h in Western blots (Fig. 1). Immunostaining was performed on smears of cauda epididymal spermatozoa. Both the anti-P26h and the anti-MBP-P26h recognized a protein on the acrosomal region of cauda epididymal spermatozoa (Fig. 2). Only acrosome-intact spermatozoa were stained by the antisera.
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To determine which germ cells express the P26h, in situ hybridization and immunostaining were performed on paraffin sections of adult testes. In situ hybridization revealed the presence of P26h mRNA in spermatocytes and round spermatids, while no signifiant labeling could be seen in late spermatids, spermatogonia, Sertoli cells, and Leydig cells (Fig. 3). Immunostaining showed that P26h is present in the cytoplasm of round spermatids and elongated spermatids and becomes almost undetectable in differentiated spermatozoa. In stage I, the immunostaining reaction was detected only in the cytoplasm of late spermatids at step 14 of spermiogenesis. No labeling was observed in any other cells of the seminiferous tubules, including spermatogonia, spermatocytes, early spermatids, and Sertoli cells. The interstitial tissue, including the Leydig cells, was also negative (Fig. 4A). Similar results were observed in stages II, III, and IV of the seminiferous cycle (not shown). Thus, only the cytoplasm of late spermatids was labeled in the above-mentioned stages. In stage V, the immunostaining reaction could be still detected in the scant remaining cytoplasm of the elongated spermatids, while no other cells were labeled (Fig. 4B). In stage VI, while the cytoplasmic immunostaining reaction of the late spermatids is now hardly detected, a weak but definite staining reaction is observed in the cytoplasm of early spermatids that are at step VI of spermiogenesis (Fig. 4C). The intensity of this weak staining reaction gradually increases in stage VII in the cytoplasm of step 7 spermatids (Fig. 4D) and in steps 8 (Fig. 5A), 9 (Fig. 5B), 10 (not shown), 11 (Fig. 5C), and 12 (not shown) of spermiogenesis. The staining reaction intensity then reached a peak in the cytoplasm of step 13 spermatids (Fig. 5D). P26h was undetectable on differentiated spermatozoa in the testis (Fig. 4).
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Figure 6 shows typical results of three independent experiments of Northern blot analyses performed on total RNA from hamster testes of 1–15 wk of age probed with both P26h and actin cDNA probes labeled with 32P. P26h transcripts were undetectable at 1 wk of age and first appeared in the testes of hamsters at 3 wk of age. This P26h transcript expression increased until it reached a maximum intensity at 5 wk of age and remained constant throughout adulthood. From 3 wk of age to adulthood only one transcript of 1081 bp was detectable.
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In situ hybridization revealed the same temporal expression of P26h mRNA as for the Northern blot analyses. The DIG-labeled cRNA probe was used to detect P26h transcripts. No transcript was detected in testes of hamsters at 1 wk of age (not shown), whereas at 3 wk, P26h transcripts were detected in spermatocytes of several seminiferous tubules (Fig. 7A). P26h transcripts in testes of adult animals were also detected in spermatocytes and round spermatids, and in this case, the signal intensity was higher than the signal intensity characterizing testes of 3-wk-old males (not shown). A great variability in labeling intensity was observed among seminiferous tubules within a testis of 3-wk-old males (Fig. 7A); the most developed tubules were the most heavily labeled. Immunostaining of testis sections from testes of 4-wk-old males revealed the presence of P26h in the cytoplasm of round spermatids (Fig. 7B).
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DISCUSSION
P26h was first described by our laboratory as a hamster sperm protein showing species-specific affinity for zona pellucida glycoproteins [18]. P26h has been shown to be acquired by spermatozoa during epididymal transit [1, 21] and to be glycosyl phosphatidylinositol (GPI)-anchored to the sperm surface covering the acrosome [22]. Recently, we described that P26h mRNA and the protein are strongly expressed in testes of sexually mature hamsters, and that P26h mRNA was also expressed in the epididymis at a much lower level [6]. As P26h is acquired by spermatozoa during epididymal transit [21], the cellular origin of P26h localized on the plasmalemma covering the acrosomal cap of cauda epididymal spermatozoa is puzzling.
To characterize testicular P26h, we used two different antisera. One antiserum was raised against P26h purified from proteins extracted from distal cauda epididymal spermatozoa and the other against the recombinant fusion protein MBP-P26h produced with a cDNA cloned from a testicular cDNA library. Both antisera recognized a P26h band on Western blots of sperm proteins extracted from cauda epididymal spermatozoa and a 65-kDa band on Western blots against the fusion protein, MBP-P26h. Moreover, during immunostaining, both antisera stain the acrosomal cap of cauda epididymal spermatozoa and a cytoplasmic protein in round and elongated spermatids in testicular sections. These results clearly demonstrate that both antibodies recognized the same protein in both the testis and on cauda epididymal sperm. Furthermore, N-terminal sequencing and peptide analysis following partial proteolysis of P26h purified from cauda epididymal spermatozoa confirm the homology between the testicular and epididymal forms of P26h, and Western blot analyses of testicular and cauda epididymal sperm proteins exhibit identical electrophoretic mobility [1, 6]. These results strongly suggest that the cauda epididymal sperm P26h and the testicular P26h are the same protein.
In this study, in situ hybridization revealed that P26h mRNAs are expressed in spermatocytes and round spermatids in the testis while immunostaining showed the presence of P26h as a cytoplasmic protein in the round spermatids and elongated spermatids, appearing at stage VI in round spermatids and increasing until stage XIII. Thus, we observe a delay between the transcript expression and its translation, suggesting that a downregulation of P26h mRNA translation occurs in spermatocytes and early spermatids (steps 1–5), and an upregulation of P26h mRNA translation occurs thereafter in round spermatids. Moreover, immunostaining revealed that P26h expression corresponds approximately with the onset of spermatid elongation [9]. Based on these results, we conclude that P26h expression is dependent on the germ cell types and the cell cycle of the epithelium.
In golden hamsters, the first mature A-spermatogonia appears at Day 12 postpartum and the first round of spermatogenesis is completed at Day 36. Thus, the whole initial cycle of spermatogenesis lasts approximately 26 days. Throughout this first round, each germ cell type will appear successively following their temporal appearance. The sequence of this temporal appearance follows the same developmental course of germ cells that is also characteristic of adult spermatogenesis [23]. Then, with regard to our results, we expected that the appearance of P26h mRNA will correspond with the first appearance of spermatocytes while P26h appearance will correspond with the first appearance of step 6 round spermatids. Northern blots revealed that the P26h transcripts first appear on Day 21. This age corresponds with the presence of spermatocytes in immature tubules [23]. In situ hybridization of 21-day-old hamster testis confirmed the presence of P26h mRNA in spermatocytes. Immunostaining of 28-day-old hamster testis confirmed the first appearance of P26h in round spermatids. These data also support the idea of translation regulation in spermatocytes and round spermatids.
As mentioned above, the cellular origin of P26h on the acrosomal cap of cauda epididymal sperm is puzzling. Indeed, Sertoli cells are the principal cells in the seminiferous tubules with secretory functions [24], and few publications relate protein release by the germ cells [25]. Our results suggest that P26h can be released by the haploid testicular germ cells, thereafter brought to the epididymis, where it binds to the sperm surface covering the acrosomal cap. Several lines of evidence support this hypothesis. First, P26h on epididymal sperm and testicular P26h appear to be the same protein. However, testicular P26h and epididymal P26h differ by their localization. Indeed, testicular P26h is a soluble cytoplasmic protein, whereas epididymal P26h is GPI-anchored. This means that P26h is subject to modification throughout epididymal transit, allowing binding of P26h to the sperm head. Moreover, weak expression of P26h mRNA in the epididymis leads to the conclusion that epididymal P26h originates from the testis. Indeed, P26h mRNA is expressed strongly in the testis and at a much lower level in the corpus epididymis [6]. Thus, two possibilities exist regarding the origin of P26h in the epididymis: first, that P26h is released by the germ cells and carried thereafter to the epididymis; and second, that P26h is secreted by the principal cells of the epididymis of the corpus segment. We have previously shown that the highest P26h concentration is found in the fluid of the epididymal initial segment and that the amount of P26h progressively decreases along the epididymis as it accumulates on the sperm surface [1]. Thus, the highest P26h concentrations characterize the fluid of the proximal epididymal segment, while epididymal P26h mRNA is detectable in low concentrations only in the corpus segment. These observations favor the hypothesis that P26h added to the sperm surface during epididymal transit originates from the testis. A dual origin for P26h, testicular and epididymal, is another possibility. If this is the case, the majority of P26h would be of testicular origin, considering the low levels of P26h transcript present in the principal cells of the corpus epididymidis [6]. The function of the corpus epididymal P26h mRNA in sperm maturation remains to be elucidated.
The mechanism by which P26h reaches the epididymis is still under investigation. P26h could be transported via proteosome-like-particules, via seminiferous tubule fluid as a soluble protein, associated with the cytoplasmic droplet, or following a pathway such as the SGP-2 protein. This protein is secreted by the Sertoli cells, endocytosed in the rete testis, and resecreted in the caput epididymis [26]. It is apparent that the release of P26h by germ cells followed by its addition to the epididymal sperm surface represents a nonclassical pathway.
Depending on its localization, P26h could be a protein that possesses two different functions: one within the germ cell and another when associated with cauda epididymal spermatozoa. Indeed, involvement of epididymal P26h in the event of fertilization has been well documented [2], but the mechanism by which P26h interacts with the zona pellucida is still unknown. In this regard, it is interesting to note that carbonyl reductase-specific inhibitors inhibit hamster sperm-zona pellucida binding in vitro (unpublished data). Within the cell, it has been described recently that recombinant P26h, produced with testis cDNA, possesses enzymatic activity such as NAD+-dependent secondary alcohol dehydrogenase activity and an affinity toward steroids [27]. This suggests that P26h could perform an enzymatic function during spermatid elongation. P26h could thus be considered a "moonlighting" protein. Moonlighting proteins have been recently described as proteins that possess multiple functions and different subcellular localizations. Many of these proteins have been described in different biological systems [28], such as an extraribosomal proteins that exhibit extraribosomal functions [29], an ATP-dependent protease that also chaperones protein biogenesis [30], and the PutA, a membrane-associated proline dehydrogenase that plays a transcriptional repressor function when localized in the cytoplasm [31, 32]. In mammalian spermatozoa, the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) exists in two forms, one as a soluble peroxidase in spermatids and also as a structural protein on the tail midpiece of the fully differentiated spermatozoa [33]. Thus, depending upon its localization within or outside the cell, P26h could play multiple roles as do other moonlighting proteins. P26h may have alcohol dehydrogenase activity in the testis that is possibly involved in the elongation of round spermatids, and secondly, it may be a protein that is released from haploid germ cells of the testis, binds the sperm during epididymal transit [1, 6, 21], and plays a role during fertilization [2].
In conclusion, P26h appears in round and elongated spermatids in the golden hamster, disappears in differentiated spermatozoa, and then reappears on spermatozoa during epididymal maturation (Fig. 8). This is an uncommon mechanism allowing sperm to acquire protein, with the mechanism being the release of P26h by haploid germ cells, its transport to the epididymis, and its acquisition by sperm during epididymal transit.
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ACKNOWLEDGMENTS
We thank Dr. Pierre Etongue Mayer for valuable advice regarding the fusion protein production and Dr. Janice Bailey for valuable criticism.
FOOTNOTES
First decision: 26 December 2000.
1 This work was supported by a Medical Research Council of Canada grant to R.S. 
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
Accepted: February 12, 2001.
Received: November 30, 2000.
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