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BOR - Papers in Press, published online ahead of print July 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026849
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BIOLOGY OF REPRODUCTION 71, 1879–1889 (2004)
DOI: 10.1095/biolreprod.103.026849
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Localization of the Chaperone Proteins GRP78 and HSP60 on the Luminal Surface of Bovine Oviduct Epithelial Cells and Their Association with Spermatozoa1

M. Boilard3, C. Reyes-Moreno3, C. Lachance4, L. Massicotte3, J.L. Bailey3, M.-A. Sirard3, and P. Leclerc2,4

Département des Sciences Animales,3 Département d'Obstétrique et de Gynécologie,4 Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, QC, Canada G1K 7P4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.

female reproductive tract, fertilization, gamete biology, sperm, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The spermatozoon is a highly differentiated motile cell responsible for the meeting between the paternal and maternal genome moieties leading to the creation of a new organism. In mammals, after ejaculation, the sperm cells travel through the female genital tract and undergo a variety of physiological modifications. With these changes occurring, male gametes acquire the characteristics required for fertilization. This phenomenon is known as capacitation. The uterus participates in the capacitation process through the action of uterine fluids [1] and in the sperm transport to the fertilization site by its contractions [2]. Once the uterotubal junction is crossed, spermatozoa are exposed to a new environment, the oviduct. Because the deposition of spermatozoa in the female genital tract may occur several hours before ovulation, this tube-shaped organ has a major task to accomplish in the fertilization process, which is to provide a sufficient number of live and competent spermatozoa to the ovum whenever the ovulation occurs.

In the isthmus, the bovine oviduct forms a sperm reservoir [3–5], where spermatozoa accumulate and bind to the apical plasma membrane of oviduct epithelial cells (OEC). The motility of bound and unbound sperm as well as their viability and capacitation status are influenced by the fluids produced by the oviduct and, more specifically, the proteins secreted by epithelial cells [6–15]. Recently, studies in the rabbit [16], horse [17], and bovine [18] have demonstrated that the direct contact between spermatozoa and the apical plasma membrane of OEC (OAPM) is important for the maintenance of motility and viability. As uncapacitated spermatozoa bind more strongly to the epithelium than the capacitated ones [19], it is suggested that spermatozoa bind to oviduct epithelial cells, where they are influenced by factors to undergo capacitation and are released once they acquire the characteristics required to fertilize the egg [20].

The sperm reservoir would therefore be very important to optimize the fertilization process. It has recently been shown that sperm binding to oviduct epithelial explants can be indicative of bull in-field fertility evaluated by the nonreturn rate [21]. In the bovine, the attachment between spermatozoa and OEC occurs in a Ca2+-dependent manner through the interaction between sperm lectins and oviduct epithelium fucose residues [22, 23]. However, the characterization of this sperm/oviduct interaction is not sufficient to describe the mechanism by which sperm viability, mobility, and fertilizing capacity are maintained or improved by this contact [16–18, 24, 25], and no oviductal factors and no cellular mechanisms involved in the modulation of sperm functions have been identified.

The goal of the present study was to identify oviduct-originating factors that associate with spermatozoa and putatively affect their cellular activity. As we previously demonstrated that the motility-enhancing effect of OAPM was lost when these membrane extracts were heat treated [18], the involvement of proteins was first investigated. Using a proteomic approach, we identified two proteins from the oviduct epithelial cell apical plasma membrane that remained attached to spermatozoa upon a short (30-min) exposure and two successive stringent washes through discontinued Percoll density gradients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Apical Plasma Membranes

The preparation of OAPM was done on the basis of what was previously described as we and others obtained a good enrichment of apical plasma membranes from oviduct epithelial cell using this procedure [16, 18]. Briefly, oviducts from cows in early estrus were collected at the slaughterhouse, maintained at 4°C during transport, and dissected from other tissues at the laboratory. OECs were recovered nonenzymatically by stripping the oviducts and collecting the emerging fluid, which contained the epithelial cells forming multicellular sheets of epithelium with beating cilia. The cells were directly subjected to the apical plasma membrane-enrichment procedure [16, 26] immediately after their recovery. In detail, cells from eight oviducts were homogenized with a polytron aggregate homogenizer (Kinematica, Luzern, Switzerland) in 20 ml of buffer 1 (60 mM mannitol, 5 mM EGTA, adjusted to pH 7.4 with 1 M Tris-HCl pH 7.4; all the chemicals were from Sigma Chemical Co., St. Louis, MO). Then 200 µl of 0.1 M MgCl2 was added to the homogenate, which was maintained on ice for 30 min to agglutinate the membranes of nonapical origin. A first centrifugation (3000 x g) was performed at 4°C for 15 min. The supernatant containing the apical membranes was removed and centrifuged at 27 000 x g for 30 min. The resulting supernatant was then removed and the pellet containing the membranes was resuspended in 20 ml of buffer 2 (60 mM mannitol, 7 mM EGTA, pH adjusted to 7.4 with Tris base) and homogenized with a Potter S homogenizer (Fisher Scientific, Nepean, ON, Canada). The mixture was then resubmitted to the purification steps involving incubation with MgCl2 for 30 min and centrifugation at 3000 x g and 27 000 x g as described above. The pellet was resuspended in 20 ml of buffer 3 (300 mM mannitol, pH 7.4, with 0.1 M Tris-HCl pH 7.4) and again homogenized with the Potter S. The final mixture was pelleted for the last time at 27 000 x g.

In the present study, two different types of OAPM were used. The first type was derived from freshly extracted OEC (fOAPM) and the second type was derived from cultured OEC (cOAPM). Both types of apical membrane extracts have been shown to positively affect sperm motility [18] and no difference was noticed between fOAPM and cOAPM. The cOAPM were prepared as follows: OECs were recovered as described above and washed by three successive sedimentations in Hanks medium (137 mM NaCl, 5 mM KCl, 4.5 mM NaHCO3, 1.1 mM Na2HPO4, 400 µM KH2PO4, 5.5 mM D-Glucose, 5 mM PIPES, pH 7.4 with NaOH) containing 5% fetal bovine serum (FBS; Medicorp, Montréal, QC, Canada) and cultured at 38.5°C and 5% CO2 in TCM 199 (Earle salts; Invitrogen, Burlington, ON, Canada) supplemented with 10% calf bovine serum (ICN, Costa Mesa, CA), 0.2 mM pyruvate, and 50 µg/ml gentamycin. After 16 h of incubation, OEC formed free-swimming vesicles with apical beating cilia on the outer surface, as also reported by Walter [27]. As reported [27], these vesicles that stay in suspension during culture, remain differentiated for a long period (over 12 days), while those attaching to the plastic dish underwent dedifferentiation quite rapidly. This emphasizes the fact that both cOAPM and fOAPM are very similar to each other. The vesicles were separated from the culture media by a 50 x g centrifugation for 2 min. The cOAPM were obtained by running the apical plasma membrane enrichment protocol described above on these cultured cells except that the first 27 000 x g pellet was homogenized directly into buffer 3 and the apical material was pelleted again at 27 000 x g instead of going through a second step of purification with buffer 2. Protein concentration in each preparation was determined by the BCA protein assay (Pierce, Rockford, IL) after solubilization of TCA-precipitated OAPM proteins. The amount of OAPM used in any experiment was determined according to their protein content.

Preparation of Radiolabeled OEC

The OEC were prepared and cultured as described above for the preparation of cOAPM. All incubations required for labeling were done at 38.5°C and 5% CO2. The cells were resuspended in 70 ml of RPMI 1640 medium (ICN) and incubated for 15 min in a 75-cm2 culture flask. They were then washed again by a 2-min centrifugation at 50 x g. The final pellet was resuspended in RPMI 1640 containing 1% FBS (Medicorp) and 50 µCi/ml of radioactive amino acids (Tran35S-label; ICN) and incubated for 3.5 h for radiolabeling. The cells were then washed by centrifugation and a sample of radiolabeled OEC was dissolved into 50 µl of 1-D electrophoresis loading buffer (125 mM Tris-HCl, pH 6.8, 4.6% SDS, 20% glycerol, 87 µM bromophenol blue, 10% ß-mercaptoethanol) or with 250 µl of two-dimensional (2-D) electrophoresis loading buffer (8 M urea, 2% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfate), 0.5% IPG buffer for pH 3–10 linear isofocusing; Amersham Pharmacia Biotech, Piscataway, NJ). Washed, radiolabeled OECs were also used to prepare 35S-labeled cOAPM.

Sperm Preparation and Treatment

In the present study, both fresh and frozen bull semen was kindly donated by the Centre d'Insémination Artificielle du Québec (CIAQ Inc., St-Hyacinthe, QC, Canada). For each experiment, straws containing frozen pooled semen from five bulls were thawed in a water bath at 37°C for 1 min. The semen was washed twice at 250 x g for 10 min using a modified Tyrode medium supplemented with BSA (fraction V), pyruvic acid, and gentamycin (Sp-TALP; [28]). Sperm concentration and motility was determined using a computer-assisted semen analyzer (CASA) (Hamilton Thorne Research, version 12.0f). In the first set of experiments, aliquots equivalent to 5 x 106 motile cells were added to each of six tubes containing 500 µl of Sp-TALP and 150 µg cOAPM proteins. After 30 min of coincubation at 38.5°C and 5% CO2, the entire volume was layered on top of a discontinued 45%/60% Percoll gradient (2 ml each) and was submitted to a 30-min centrifugation (700 x g) to eliminate any material that was not strongly bound to the spermatozoa. The pellet was washed by centrifugation in 5 ml of Sp-TALP at 370 x g, layered on a second 45%/60% Percoll gradient, and centrifuged 30 min at 700 x g. After the second Percoll wash, sperm were washed twice by centrifugation in Sp-TALP medium. Final sperm concentration was determined and adjusted to 25 x 106/ml. Motility parameters were assessed using CASA after 0, 2, 6, and 12 h of incubation at 38.5°C and 5% CO2. At the same time, the acrosomal integrity and viability were evaluated by eosin-nigrosin staining [29]. The motility, viability, and acrosome integrity values were obtained by the analysis of a minimum of 100 sperm cells per treatment. This experiment was repeated three times on different days using different cOAPM preparations and different semen straws. Statistical analyses were done using the Statistical Analysis System (release 6.12; SAS Institute Inc., Cary, NC) general linear model procedure for repeated measures analysis.

Identification of Apical Plasma Membrane Proteins that Bind to Spermatozoa

Spermatozoa were prepared and incubated with radiolabeled cOAPM as described above for the unlabeled material. The sperm pellet obtained after centrifugation through the second Percoll gradient was washed twice with cold phosphate buffered saline (PBS; 0.15 M NaCl, 10 mM NaH2PO4, pH 7.4) and the proteins were solubilized in either one-dimensional (1-D) or 2-D electrophoresis loading buffer. The proteins were separated either by 1-D SDS-PAGE [30] or 2-D electrophoresis using Immobiline DryStrip gels carrying an immobilized linear 3–10 pH gradient and the IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech) for the first dimension and SDS-PAGE for the second dimension. The gels were first fixed for 30 min in a 40% methanol and 10% acetic acid solution and then soaked for 30 min in Amplify (Amersham Pharmacia Biotech) to enhance radiation. The gels were next dried and subjected to autoradiography using Kodak BioMax MR films. The mass and pI of labeled proteins bound to sperm were determined.

Unlabeled OEC proteins were separated in parallel by 2-D electrophoresis, transferred on a polyvinylidene fluoride (PVDF) membrane and stained with Coomassie brilliant blue. Pieces of PVDF membrane containing the proteins with mass and pI identical to the 35S-labeled proteins bound to spermatozoa were excised and processed for N-terminal sequencing by automatic Edman degradation (Applied Biosystems, Foster city, CA) at the Service Protéomique de l'Est du Québec (Centre Hospitalier Universitaire de Québec, QC, Canada).

To confirm that the OEC proteins identified using the procedure described above really bound to spermatozoa, their presence was investigated by Western blot procedures on proteins extracted from spermatozoa incubated for 30 min in the presence or absence of unlabeled fOAPM. After the incubation, spermatozoa were washed on two successive Percoll gradients as described above to remove weakly attached apical membrane extracts and were counted by CASA. Sperm proteins were next solubilized in 1-D electrophoresis loading buffer, subjected to 1-D SDS-PAGE, and electrotransferred onto nitrocellulose membranes [31]. Nonspecific binding sites on the membrane were blocked using Tris-buffered saline containing Tween 20 (TBST; 154 mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.1% Tween 20) supplemented with 5% (w/v) dry skimmed milk for 1 h. After three washes in TBST, the membranes were incubated for 1 h at room temperature with monoclonal antibodies directed against the specific proteins, based on the results obtained by protein sequencing, namely GRP78 (1: 10 000; BD Biosciences-Pharmingen, San Diego, CA), HSP60 (1:10 000), PDI, or GRP58 (Stressgen Biotechnologies, Victoria, BC, Canada). Again, the membranes were washed in TBST, then incubated for 45 min with a goat anti-mouse IgG conjugated to horseradish peroxidase. At the end, the membranes were extensively washed in TBST and immunoreactive bands were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech) and film exposure according to the manufacturer's instructions.

Immunolocalization of Endogenous Sperm HSP60

Freshly ejaculated semen was brought to the lab at 25°C within 2 h. Spermatozoa were washed by centrifugation in PBS and resuspended at a final concentration of 20 x 106 cells/ml. Droplets of 35 µl of the sperm suspension were deposited on a poly-L-lysine-coated coverslip and the cells were allowed to adhere over a 30-min period. The sperm cells were next fixed/permeabilized by covering the coverslips with 1 ml of methanol and kept at –20°C for 10 min, then allowed to dry until use. At the time of use, spermatozoa were rehydrated in PBS, then incubated with PBS added with 0.1% BSA (PBS-BSA) to block nonspecific sites on the coverslips. The spermatozoa were next incubated for 1 h at 37°C with monoclonal anti-HSP60 (1:100), washed three times with PBS-BSA, then incubated for 1 h at 37°C with a goat anti-mouse-IgG conjugated to fluorescein isothiocyanate (FITC). After extensive washes in PBS, the coverslips were mounted on slides in the presence of the antibleaching agent diazabicyclo[2, 2, 2]octane (1.5% [v/v] made in 90% glycerol). Spermatozoa were examined by epifluorescence microscopy.

Biotinylation of Apical Surface Proteins of Oviduct Epithelial Cell

The apical surface localization of the identified proteins was first investigated using biotinylated cultured OEC. As stated above, when maintained in culture, OEC form multicellular swimming vesicles with neighboring cells held together and connected by junctions and with beating cilia on their surface [27]. Although these cells do not undergo mitosis, the vesicles increase in size as the cells transport substances from the medium to their interior ([27] and our own observations). This suggests that the vesicles form structures that are closed tightly enough to prevent the biotinylation of basal surface proteins. Apical surface biotinylation, precipitation, and blot assay of the identified proteins were performed as previously described [32]. Special care was taken to ensure that our protein biotinylation procedure would discriminate between proteins expressed on the cell surface and intracellular ones. To decrease the possibility of intracellular protein biotinylation, the biotinylation process was done at 4°C for 20 min instead of the manufacturer's suggested protocol (30 min at room temperature). This protocol to determine exclusive cell surface location of chaperone proteins has been successfully used in the past, as confirmed by electron microscopy procedures [33]. Briefly, cultured OEC vesicles were rinsed three times with cold PBS and were next incubated on ice for 20 min in PBS in the presence of 1 mg Sulfo-NHS-LC-biotin (Pierce, Rockford, IL) per ml. They were next rinsed three times with cold PBS. Total protein concentration in the sample was determined using the BCA Protein Assay (Pierce). The biotinylated vesicles were homogenized in 500 µl RIPA buffer (0.15 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM Tris, pH 7.6) supplemented with protease inhibitors (17 µg/ml PMSF, 2 µg/ml leupeptin, 0.7 µg/ml pepstatin) for 30 min on ice. The samples were centrifuged 20 min at 16 000 x g to remove any cellular debris. The solubilized protein extract was next processed either for the enrichment of biotinylated OEC proteins or for immunoprecipitation of HSP60 and GRP78.

Identification of Biotinylated HSP60 and GRP78 by Immunoblotting

Two different approaches were undertaken to determine whether OEC HSP60 and GRP78 were biotinylated as an indication of their localization on the apical surface of OEC. The first attempt was to determine whether HSP60 or GRP78 were present in the group of biotinylated proteins extracted from OEC. Immobilized neutravidin on agarose beads (Pierce) were added to the lysate and the mixture was incubated overnight at 4°C. The beads were washed four times by centrifugation (1 min at 13 000 rpm in a microcentrifuge) in PBS containing protease inhibitors. Finally, the beads were resuspended in SDS-PAGE loading buffer and heated at 100°C for 10 min. The proteins were subjected to 1-D SDS-PAGE and electrotransferred onto nitrocellulose membranes. The presence of HSP60 and GRP78 within the pool of biotinylated OEC proteins was investigated by Western blot using monoclonal antibodies directed against GRP78 or HSP60 as described earlier.

Immunoprecipitation of Oviduct Epithelial Cell HSP60 and GRP78 Proteins

The second approach to determine whether HSP60 and GRP78 are localized at the surface of the OEC was to immunoprecipitate the two chaperones HSP60 and GRP78 and to evaluate whether or not they were biotinylated. After the surface biotinylation procedure of OEC, the cells were lysed in RIPA and the particulate material removed by centrifugation as described earlier. This lysate was precleared for 1 h using 1 µg of nonimmune mouse IgG (Sigma Chemical Co.) and 35 µl of protein G-coupled Sepharose beads (Amersham Pharmacia Biotech), washed, and resuspended in RIPA. The beads were next eliminated by centrifugation at 3000 x g for 3 min. One microgram of either anti-HSP60 or anti-GRP78 monoclonal antibodies was added and the samples were incubated for 2 h. Washed protein G-sepharose beads were then added and the samples were incubated for an additional 2 h. The suspensions were next centrifuged at 3000 x g for 3 min, the beads containing the immune complex were washed three times with RIPA, and finally resuspended in 1-D electrophoresis sample buffer and heated at 100°C for 5 min. The proteins were submitted to SDS-PAGE, transferred on nitrocellulose membrane, processed as for the Western blot procedure except that, after blocking the nonspecific sites, the membranes were probed with horseradish peroxidase-conjugated avidin (Pierce) to detect biotinylated proteins instead of primary and secondary antibodies. At last, as for the Western blots, the positive bands on the membrane were revealed by chemiluminescence (ECL) and film exposure.

Immunohistochemical Localization of HSP60 in the Oviduct

Oviducts from three different cows in early estrus were recovered at the slaughterhouse and brought to the lab on ice within 5 h. They were dissected free from other tissues on ice and a 1-cm cross section of the isthmus and the ampulla were cut off and fixed overnight in Bouin. They were then washed twice in distilled water for 15 min and dehydrated through consecutive increasing concentrations of ethanol baths. They were then washed three times in toluene and embedded in paraffin. On the day of use, 5 µm sections were prepared, layered onto Superfrost Plus glass slides (Fisher), and allowed to dry. The slides were dipped in toluene to remove paraffin and rehydrated through decreasing ethanol concentration baths. They were next bathed for 10 min in a 1% lithium carbonate made in 70% ethanol solution to remove residual picric acid from the Bouin solution and next immersed for 10 min in a 300 mM glycine solution to block free aldehyde sites. Endogenous tissue peroxidase activity was inhibited by treating the slides for 10 min with 3% H2O2 in methanol. The antigen retrieval step was done by plunging the slides for 10 min in 10 mM boiling sodium citrate solution (pH 6.0). The slides were next covered with PBS supplemented with 1% BSA (PBSB) for 1 h to block potential nonspecific binding sites. The slides were then incubated for 2 h with either anti-HSP60 (1:100) or anti-GRP78 (1:100) or commercial nonimmune mouse IgG as a control. Upon several washes with PBS, the tissues were covered with a biotin-coupled goat anti-mouse IgG antibody (Jackson ImmunoResearch Labs) for 1 h and, after several washes, with strepavidin congugated to horseradish peroxidase (Jackson ImmunoResearch Labs) for 30 min. The protein location was finally visualized using diaminobenzidine (Sigma) and the tissue was counterstained with Gill hematoxylin (Fisher).

Oviduct Epithelial Cell Localization of HSP60 by Indirect Immunofluorescence

Oviduct epithelial cells were prepared as described above and cultured for 5 days. The media was changed once after 2 days. Half of the vesicle suspension was washed three times in a HEPES buffered Tyrode medium (TLH) [28] supplemented with 2 mg/ml BSA (TLHB). These vesicles were next incubated for 1 h in the presence of the monoclonal anti-HSP60 or anti-GRP8 antibodies (both diluted 1:100) or a rabbit polyclonal antibody (1:100) against SERCA (sarcoplasmic/endoplasmic reticulum calcium ATPase; provided by Dr. J Lytton, University of Calgary, AB, Canada) as a control for intracellular proteins. They were next washed thrice in TLHB and incubated for 45 min with the respective FITC-conjugated secondary antibody. Finally, the vesicles were washed six more times in TLHB, placed on poly-L-lysine-coated coverslips and mounted on a glass slide.

The other half of the vesicle suspension was washed three times in TLH containing 0.1% PVP40 (TLHP), then fixed in PBS containing 3.7% formaldehyde for 10 min at room temperature. They were next rinsed three times in TLHP and permeabilized for 10 min in TLHP containing 0.2% Triton X-100. The cells were next washed three times in TLHP and three times in TLHB. The localization of either HSP60 or SERCA was performed by indirect immunofluorescence, as described for the nonpermeabilized cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modulation of Sperm Functions by Bound OAPM Proteins

The first set of experiments was to determine whether, as observed when they are continuously incubated with OAPM (Boilard et al. [18]), spermatozoa incubated for a short period (30 min) with these membrane extracts still benefit from their positive effects during a subsequent prolonged incubation in the absence of OAPM. As clearly shown in Figure 1A, a significantly higher percentage of spermatozoa kept their acrosome intact when they were preincubated for 30 min with cOAPM and further incubated for 2 or 6 h (49% vs. 27% and 46% vs. 20%, respectively). The positive effect of cOAPM on acrosomal integrity was no longer observed when the sperm incubation lasted for 12 h (30% vs. 18%). Similarly, the preincubation with cOAPM retarded the loss of sperm viability during the incubation that followed cOAPM treatment and successive Percoll washes (33% vs. 13% at 6 h; Fig. 1B).



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  		FIG. 1. Effect of sperm-associated cOAPM proteins on acrosomal integrity (A) and sperm viability (B). Washed spermatozoa were preincubated for 30 min in the absence or presence of cOAPM, washed on two consecutive discontinued Percoll gradients, and then incubated for up to 12 h in TL-sperm. Results are expressed as means ± SD of three experiments. For both parameters, the curve representing treated spermatozoa was significantly different for the incubation period spanning from 0 to 6 h (treatment effect: P < 0.025) when compared with the one for control sperm cells

The sperm motility parameters were measured after 2, 6, and 12 h of incubation following the 30-min pretreatment of sperm with cOAPM. No significant difference was observed in the percentage of motility between the cOAPM-treated and control cells. Nevertheless, the different movement parameters were analyzed for the motile spermatozoa. Of these, only the linearity and the straightness of the trajectory were influenced by the cOAPM (Fig. 2, A and B, respectively). At 12 h, the cOAPM-treated spermatozoa exhibited higher linearity (73% vs. 38%) and straightness (97% vs. 75%) than untreated cells.



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  		FIG. 2. Effect of sperm-bound OAPM proteins on motility parameters. Sperm were incubated for 30 min in the absence or presence of OAPM, washed on two consecutive discontinued Percoll gradients, and then incubated for up to 12 h in TL-sperm. Sperm movements were analyzed by CASA. Results are expressed as means ± SD of three experiments. An overall significant positive effect of the treatment by OAPM was observed on linearity (A) (P = 0.01) and straightness (B) (P = 0.06)

Identification of OAPM Sperm-Binding Proteins

As shown in Figure 3A, numerous OEC proteins were labeled with 35S-methionine and 35S-cysteine. A different pattern of protein expression was observed in the OAPM fraction (data not shown) and, of these, only few bound to spermatozoa and remained attached following two successive washes through discontinued Percoll gradients (Fig. 3B). The mass and isoelectric point (kDa/pI) of the six major 35S-labeled proteins (ImageMaster Software; Amersham) were determined (80 kDa/5.0, 75 kDa/5.8, 60 kDa/ 5.5, 55 kDa/6.5, 50 kDa/5.0, and 45 kDa/5.1; all included in boxes in Fig. 3), as well as those from five additional proteins of 160 kDa/5.5, 60 kDa/6.2, 40 kDa/7.3, 37 kDa/ 7.3, 30 kDa/4.9 that showed significant binding to the sperm cells.



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  		FIG. 3. Autoradiography of 2-D electrophoretic patterns of 35S-radiolabeled proteins from oviduct epithelial cells (A) and from sperm incubated with radiolabeled apical membrane extracts (B). Two-dimensional electrophoretic pattern of unlabeled oviduct epithelial cell proteins stained with Coomassie brilliant-blue is also shown (C). Proteins chosen for N-terminal amino-acid sequencing are included in boxes

The proteins spots corresponding to the mass and pI determined for the six most labeled proteins bound to spermatozoa were excised from the PVDF membrane containing the 2-D gel separated proteins extracted from OEC and submitted for N-terminal sequencing. Of these, three were successfully identified (Table 1). The 80-kDa protein was identified as the glucose-regulated protein 78 (GRP78), also known as immunoglobulin heavy chain binding protein (BiP). Its identity was confirmed by Western blots using a commercial monoclonal antibody on total OEC proteins resolved by 2-D gel electrophoresis (not shown). Similarly, the 60-kDa protein with a pI of 5.5 was identified as the heat shock protein 60 (HSP60), the identity of which was also confirmed by Western blots using a commercial monoclonal antibody (not shown). Finally, the N-terminal amino acid sequence of the 55 kDa/6.5 pI protein matched to some extent to the glucose-regulated protein 58 (GRP58), also known as protein disulfide isomerase (PDI). However, its identity could not be confirmed by Western blot using the different antibodies against GRP58 (anti-PDI donated by D. Ferrari, Max Plank Institute for Biochemical Research, Gottingen, Germany; Anti-PDI and Anti-ERP57; Stressgen Biotechnologies). It has to be remembered, however, that 13 of the 15 amino acids determined in the N-terminus shared identity with GRP58 (Table 1).


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  		TABLE 1. N-terminal sequences obtained from the 2-D gel-excised pro teins

The binding of OAPM-originating HSP60 and GRP78 to spermatozoa was next assessed using commercial antibodies upon sperm incubation with fOAPM and washes through two consecutive Percoll gradients. As shown in Figure 4A, a strong GRP78 signal is observed in the proteins extracted from spermatozoa previously incubated with fOAPM, whereas it is not detected when spermatozoa are incubated in the absence of fOAPM, confirming that GRP78 was acquired by sperm during the coincubation period with fOAPM. Unlike GRP78, the presence of HSP60 was already in spermatozoa incubated in the absence of fOAPM (Fig. 4C). It was detected in both epididymal and freshly ejaculated spermatozoa but not in the egg yolk-containing semen extender used for cryopreservation (data not shown), indicating that it is either an endogenous protein or a protein added during the sperm transit through the epididymis or ejaculatory duct. However, as for GRP78, a higher level of HSP60 was detected when the sperm cells were incubated with fOAPM, confirming that HSP60 is also acquired by spermatozoa during their incubation with fOAPM.



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  		FIG. 4. Immunodetection of GRP78 and HSP60 in bull spermatozoa incubated with fOAPM. Washed cryopreserved spermatozoa were incubated for 30 min in washing medium in the absence (lane 1) or presence (lane 2) of fOAPM. After the incubation, spermatozoa were washed two more times on Percoll gradients, as described in Materials and Methods, and their concentration finally evaluated by CASA. Proteins extracted from equal numbers of washed cryopreserved spermatozoa were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-GRP78 (A, B) or anti-HSP60 (C, D) monoclonal antibodies. A, C) The signal observed using anti-GRP78 and anti-HSP60, respectively, and their respective Ponceau red-stained nitrocellulose membrane (B, D) as an indication of the amount of sperm proteins subjected to the Western blot procedure. This figure is representative of three independent experiments. The position of molecular weight standards (kDa) is indicated on the left

Immunolocalization of HSP60 in Mature Spermatozoa

As the presence of HSP60 has never been reported in mature spermatozoa, the localization of this chaperone protein was investigated in freshly ejaculated bovine spermatozoa. Using an indirect immunofluorescence approach, a significant and specific labeling was observed in the midpiece of fixed/permeabilized spermatozoa (Fig. 5A). Identical results were obtained with cauda epididymal spermatozoa (not shown), suggesting that this protein is endogenous to the sperm cell and not acquired during the epididymal transit.



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  		FIG. 5. Localization of HSP60 on mature bull spermatozoa by indirect immunofluorescence. Washed ejaculated bull spermatozoa were fixed/permeabilized with methanol, then probed with a commercial monoclonal antibody against HSP60 (A) or nonimmune mouse IgG as a control (C). A FITC-conjugated secondary antibody was then used to determine the sperm localization of HSP60. B, D) The phase contrast fields of A and C, respectively. All the panels are at x1000 magnification

Immunohistochemical Localization of HSP60 in the Bovine Oviduct

Although HSP60 and GRP78 are ubiquitous chaperone proteins, we first wanted to determine whether they are present in the cow oviduct epithelium and both the isthmus (sperm reservoir) and ampulla (fertilization site). Although a strong signal was obtained by Western blot procedures, our commercial monoclonal GRP78 antibody could not detect anything by immunohistochemical methods. On the other hand, a strong HSP60 signal was detected in the isthmus, this protein being more abundant in the epithelium than in the stroma (Fig. 6). Moreover, within the epithelial cells, a stronger labeling was observed at the apical side than at the baso-lateral sides of the cells. Similar results were obtained in cross sections of the ampulla (data not shown).



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  		FIG. 6. Localization of HSP60 in bovine oviduct. Cross sections of the oviduct isthmic regions were processed for immunohistochemistry using an HSP60 monoclonal antibody (A, C) or a nonimmune mouse IgG (B). The presence of the protein was revealed using a secondary antibody coupled to biotin, horseradish peroxidase-conjugated avidin-peroxidase, and diaminobenzidine

Apical Surface Localization of HSP60 and GRP78

Because GRP78 and HSP60 are known to be expressed mostly in the endoplasmic reticulum and mitochondria, respectively, both organelles being unreachable by spermatozoa in vivo, it was important to determine whether or not these proteins are localized at the apical (luminal) surface of OEC. Our first attempt was to determine whether HSP60 and GRP78 are present among the OEC surface biotinylated proteins. Total biotinylated proteins were recovered using neutravidin-conjugated agarose beads and, after SDS-PAGE and transfer onto nitrocellulose membrane, both HSP60 and GRP78 were detected (Fig. 7).



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  		FIG. 7. Identification of HSP60 and GRP78 within the surface biotinylated proteins of oviduct epithelial cells. Proteins recovered from biotinylated (Biot) or unbiotinylated (UB) cultured oviduct epithelial cells were solubilized in RIPA buffer, collected using neutravidin-conjugated beads, and processed for Western blotting using anti-HSP60 and anti-GRP78 monoclonal antibodies. This figure is representative of three independent experiments. The position of molecular weight standards (kDa) is indicated on the left

Conversely, total protein extracts from cell-surface biotinylated OEC were immunoprecipitated using anti-GRP78 or anti-HSP60 monoclonal antibodies. The biotinylation status of the immunoprecipitated proteins was assessed on SDS-PAGE and transfer using peroxidase-conjugated avidin. Anti-GRP78 monoclonal antibody immunoprecipitated a biotinylated 78-kDa protein (Fig. 8A, left side), which proved to be GRP78, as confirmed by Western blotting on the same membrane using the anti-GRP78 monoclonal antibody (Fig. 8A, right side). Other proteins that coimmunoprecipitate with GRP78 also appeared to be biotinylated (p97 and p64). In addition, another protein, {approx}57 kDa, was observed in both the preclear and immunoprecipitated materials using either anti-GRP78 or anti-HSP60 lanes (left panels of Fig. 8, A and B, respectively), suggesting that this protein binds nonspecifically to the protein G-coupled Sepharose used to precipitate the immune complex. When the immunoprecipitation was performed with the HSP60 antibody, a faint 60-kDa biotinylated protein was detected, which appeared to be HSP60 (Fig. 8B, compare the left and right panels). As shown in the right panel of Figure 8B, the biotinylation process interfered with the detection of HSP60 by our commercial monoclonal antibody. Previous results in our laboratory demonstrated that our HSP60 antibody was less effective when the Western blot was performed on biotinylated apical plasma membrane proteins (data not shown). Consequently, the small amount of HSP60 immunoprecipitated from the lysate of biotinylated cells as compared with unbiotinylated cells also suggests that HSP60 was biotinylated. Taken together, these results stongly suggest that both GRP78 and HSP60 are biotinylated and, therefore, present on the luminal (apical) surface of the OEC, an area reachable by spermatozoa.



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  		FIG. 8. Identification of HSP60 and GRP78 as biotinylated proteins of OEC plasma membrane. Upon OEC surface biotinylation and protein solubilization in RIPA buffer, GRP78 and HSP60 were immunoprecipitated. The proteins were next subjected to SDS-PAGE, transferred onto nitrocellulose, and probed with avidin-HRP to determine whether or not GRP78 and HSP60 were biotinylated (left panel of A and B, respectively). The membranes were next reprobed (right panel of A and B) by Western blot using anti-GRP78 or anti-HSP60 monoclonal antibodies to confirm the presence of the proteins in sample from biotinylated (Biot) and unbiotinylated (UB) cells. IP, Immunoprecipitated proteins; PC, the proteins that bound nonspecifically to the nonimmune mouse IgG and protein G Sepharose during the preclear step of the immunoprecipitation procedure. The position of GRP78 and HSP60 are indicated by arrows in panels A and B, respectively. This figure is representative of three independent experiments. The position of molecular weight standards (kDa) is indicated on the left

Cell Surface Localization of HSP60 by Indirect Immunofluorescence

The surface expression of HSP60 and GRP78 on OEC vesicles was also investigated by indirect immunofluorescence. As for the immunohistochemistry performed on oviduct cross sections, the GRP78 monoclonal antibody was inefficient in these indirect immunofluorescence experiments. As shown in Figure 9A, HSP60 was detected on intact nonpermeabilized OEC vesicles. To ensure that only surface proteins could be detected, an antibody against a well-known intracellular protein (SERCA) was used. No signal was detected on the OEC vesicles using the SERCA antibody unless the vesicles were previously permeabilized with Triton X-100 (Fig. 9E), strengthening our results showing that HSP60 is present on the surface of OEC.



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  		FIG. 9. Localization of HSP60 on the surface of oviduct epithelial cells. Nonpermeabilized (NP) epithelial vesicles were incubated with anti-HSP60 monoclonal antibody (A) or with commercial mouse IgG (B) then revealed with an anti-mouse IgG coupled to FITC. Indirect immunofluorescence was also performed using an antibody directed against an intracellular protein, the SERCA, which failed to detect the antigen in nonpermeabilized vesicles (C), whereas a positive signal was observed in permeabilized (P) cells (E). Signal obtained with nonimmune rabbit serum on nonpermeabilized (D) or permeabilized (F) vesicles is also shown


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In 1951, Austin and Chang simultaneously and independently demonstrated the necessity for mammalian sperm to spend some time in the female genital tract to become fertilization competent [34, 35]. Different proteins secreted by oviduct epithelial cells in vivo or in vitro interact with sperm, bind to them, and sometimes improve their fertilizing potential [6–12, 14]. A coincubation system using partially purified apical plasma membranes from OEC (OAPM) was set up to better characterize the interactions between spermatozoa and these cells without the influence of their secretions. Enriched apical membrane extracts from bovine oviduct epithelium were produced and confirmed by enzymatic assays as described in a previous manuscript [18]. Using such a system, it was demonstrated that the apical fraction of OEC contains proteic factors that modulate sperm motility and intracellular calcium concentration ([Ca2+]i), whereas extracts from mammary gland cells do not [18].

In the present study, we clearly demonstrate that a 30-min preincubation with cOAPM was beneficial to spermatozoa as revealed by the viability, acrosomal integrity, and sperm movements evaluation after several hours of incubation without those OAPM. However, although not investigated here, it is possible that apical plasma membrane factors other than GRP78 or HSP60 that did not bind strongly enough to spermatozoa triggered intracellular mechanisms during the coincubation with apical membrane extracts and positively affected sperm functions a posteriori. This issue remains to be investigated in the future. In the present study, although the percentage of sperm motility was not affected by the short exposure to the OAPM, the motile cells expressed higher linearity and straightness after 12 h of incubation when they were preincubated for 30 min in the presence of the OAPM. Low linearity and straightness of sperm movement are associated with fertility problems in humans [36–39]. Hyperactivation is a type of motility with low linearity and straightness [40], which is generally associated with sperm capacitation and which requires high [Ca2+]i. Therefore, our results showing a sustained sperm linearity and straightness caused by preincubation with cOAPM are in total agreement with our previous report, where fOAPM was shown to maintain low [Ca2+]i [18]. Moreover, high [Ca2+]i are also required for spermatozoa to undergo the acrosome reaction. In the present study, it was demonstrated that a higher proportion of spermatozoa maintained the integrity of their acrosomes when they were preincubated in the presence of cOAPM proteins (Fig. 1), which, again, agrees perfectly with the lower [Ca2+]i previously reported [18].

In addition to the proteins described here, many proteins might have associated with sperm, without being detected by autoradiography because of the inherent limits of the protein analysis approaches used. The 35S radiolabeling technique allows only the study of proteins synthesized during the labeling incubation period and not those that were already present at the membrane before the labeling. In addition, many synthesized proteins might have been unobservable because of their low abundance or because of their low methionine or cysteine content, thus precluding 35S radiolabeling. Nevertheless, many associated proteins were detectable. Three of the six major proteins associated with spermatozoa tightly enough to resist the washing procedure were identified. The identities of GRP78 and HSP60 were confirmed using commercial antibodies and Western blot on 2-D gel separated proteins. Although the mass and isoelectric point of the third N-terminal sequenced protein corresponds to those already determined for GRP58 in the hamster [41], none of the three different antibodies tested succeeded in confirming that our gel-excised and sequenced protein was effectively GRP58. As 13 out of 15 amino acids obtained in the N-terminal sequence showed identity to human GRP58 (Table 1), it has to be determined whether our lack of success with the different antibodies comes from species specificity of the antibody or from the identification of a close, although distinct, relative of GRP58. However, GRP58 cannot be eliminated as a potential sperm function modulator but was not investigated further in our study.

Because the proteins identified were excised from a gel containing all OEC proteins to ensure a larger quantity of proteins (Fig. 3C), it was important to confirm the binding of GRP78 and HSP60 to sperm by immunoblot (Fig. 4) to eliminate the possibility that a protein showing no affinity to sperm could have been sequenced due to the presence of many proteins with similar pI and molecular weight. In fact, whether GRP78 or HSP60 directly interacts with a sperm element or whether these proteins are associated with other proteins responsible of the binding remains elusive. Our results clearly show that no GRP78 is detected in bovine sperm before incubation with OAPM, whereas it is easily detectable upon sperm exposure to apical plasma membrane of OEC. On the other hand, although HSP60 was already present in spermatozoa, its levels in sperm protein extracts increased upon sperm incubation with OAPM, suggesting that it was also acquired from OAPM. The present study represents the first evidence that HSP60 is present in fully mature ejaculated spermatozoa. Moreover, using an indirect immunofluorescence approach, our results clearly show that HSP60 is located in the midpiece of mature bull ejaculated spermatozoa. Previous studies have shown that HSP60 is localized in the somatic type of mitochondria in both spermatogonia and primary spermatocytes [42, 43]. The spermatocytes that are more advanced in the spermatogenetic process have HSP60-depleted condensed mitochondria. It is not known, at the present time, whether the presence of HSP60 in mature bull spermatozoa is a matter of species specificity.

However, because both GRP78 and HSP60 are proteins known to be important intracellular proteins implicated in a wide variety of functions [44–47], their presence in the apical extract preparation was rather surprising and needed to be clearly demonstrated. Using an immunohistochemical approach, it was found that HSP60 is highly abundant at the apical side of epithelial cells, which is in complete agreement with the presence of this chaperone protein in the apical membrane extracts (present manuscript). On the other hand, these results were not sufficient to determine whether or not HSP60 is present at the luminal face of the epithelial cells and, therefore, available to sperm binding. Although the commercial GRP78 antibody detects the protein in cell or apical membrane extracts (present manuscript), no result could be obtained by immunohistochemical methods. Therefore, to assess whether this protein is present at the cell surface of the oviduct epithelium, additional experiments using cell surface biotinylation were necessary to validate this hypothesis. In the present study, both HSP60 and GRP78 were detected within the pool of OEC surface biotinylated proteins and, conversely, surface biotinylated proteins of the appropriate mass were detected among the immunoprecipitated HSP60 or GRP78, respectively. These results confirmed that both HSP60 and GRP78 are present on the oviduct epithelial cell surface.

Although not a general feature, the surface localization of the chaperone HSP60 and GRP78 has been previously reported. Soltys and Gupta [33] demonstrated the cell surface expression of HSP60 in immortalized Chinese hamster ovary cells [33] and no relation was made with any particular cellular or biochemical mechanism. In addition, the expression of GRP78 on the cell surface of different cancer cell lines has also been reported [48–51]. According to the amino acid sequence of human GRP78, this protein contains a signal peptide and might be expressed extracellularly with one putative transmembrane domain. However, these characteristics were absent when considering the human HSP60 amino acid sequence, even though the surface location of this chaperonin has been demonstrated [33]. In contrast with these previous studies, the results presented here were obtained using freshly extracted cells and on primary cell culture. Our data represent new evidence that, at least in the oviduct, normal or untransformed epithelial cells express HSP60 and GRP78 on their surface under physiologically relevant conditions.

It is well established that HSP60 and GRP78 protein expression is enhanced by cellular stresses. Therefore, one could argue that the surface expression of HSP60 and GRP78 was induced by the exposure of the oviducts to stresses occurring during the isolation and culture of OEC vesicles. However, OEC are routinely prepared and used to assist the in vitro culture of in vitro fertilized eggs in our laboratory [52] and are also routinely known to maintain synchronized ciliary activity for days if not weeks, suggesting that these culture conditions they are subjected to are not detrimental. In addition, it has been reported in an independent study that these swimming vesicles maintained their state of differentiation for more than 10 days in culture [27].

The results presented in this study suggest that the protective effect of OAPM on spermatozoa is caused by the binding of OAPM proteins to the sperm cells or by a translocation of proteins from OAPM to the sperm plasma membrane. However, the effect of those proteins on sperm remains to be determined. In the present study, among all the OAPM proteins that might bind to spermatozoa, we identified two proteins, HSP60 and GRP78, that strongly bind to spermatozoa upon a short, 30-min, sperm/OAPM coincubation. More investigations are required, however, to determine whether these two ubiquitous proteins affect sperm functions and what mechanisms are involved. In addition, as HSP60 is already present in bull spermatozoa, one can ask what the relevance is of this protein in the beneficial effects of OAPM on sperm cells. Our results show that endogenous HSP60 is located in sperm mitochondria, in the midpiece. However, further studies are needed to determine where exogenous (OEC) HSP60 binds to bull spermatozoa. This information would be helpful to determine whether this exogenous chaperonin affect sperm functions and to investigate the mechanisms involved. In normal conditions, molecular chaperones participate in various functions, such as correct protein folding, protein transport across the membranes of organelles, formation of oligomeric protein complexes, organelle biogenesis (reviewed in [44–47, 53–57]). Therefore, one can speculate that, during the sperm incubation with apical membrane extracts, these molecular chaperone proteins participate in the protection of the integrity of sperm plasma membrane by interacting with the proteins or by transferring important proteins from the oviduct epithelial cell membranes to the sperm plasma membrane, which might affect sperm signaling pathways. The involvement of some molecular chaperones in different signaling pathways, such as cell cycle control [58], steroid receptor signaling [59], and the regulation of apoptosis [60], has been reported.

This study clearly demonstrates that both HSP60 and GRP78 are expressed on the surface of oviduct epithelial cells and they strongly associate with spermatozoa. However, further studies need to be performed to determine whether these proteins are involved in the maintenance of sperm viability and integrity within the oviduct sperm reservoir or whether they are involved in the capacitation process where spermatozoa become competent at fertilizing the egg.


   ACKNOWLEDGMENTS
 
Fresh and frozen sperm samples were graciously provided by L'Alliance Semex Inc. (Guelph, ON, Canada) and the Centre d'Insémination Artificielle du Québec (St-Hyacinthe, QC, Canada). The authors wish to express special thanks to Dr. D. Ferrari from Max Plank Institute for Biochemical Research, Gottingen, Germany, who graciously shared his PDI antibody with us. Thanks also to F. Giguère for her help in oviduct dissection, S. Goupil for his help with immunoprecipitation procedures, and Dr J. Lytton (University of Calgary, AB, Canada) for his gift of anti-SERCA antibody.


   FOOTNOTES
 
1 Supported by the Natural Science and Engineering Research Council of Canada, L'Alliance SEMEX, and the Fonds Québécois de la Recherche sur la Nature et les Technologies. M.-A.S. and P.L. contributed equally to this work. Back

2 Correspondence: Pierre Leclerc, Endocrinologie de la reproduction, Pav. St-François d'Assise, 10, de l'Espinay, Québec, QC, Canada G1L 3L5. FAX: 418 525 4195; Pierre.Leclerc{at}crsfa.ulaval.ca Back

Received: 22 December 2003.

First decision: 16 January 2004.

Accepted: 22 July 2004.


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 DISCUSSION
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