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Regular Article |
a Pest Animal Control Cooperative Research Centre, CSIRO Sustainable Ecosystems, Canberra, ACT 2601, Australia
b Department of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gamete biology, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature New Zealand White rabbits (>6 mo old) and Balb-C mice (>8 wk old) were obtained from breeding colonies housed at CSIRO, Division of Wildlife and Ecology, and studied with the approval of the Institute's Animal Care and Ethics Committee. Animals were maintained under a light regime of 16L:8D and were supplied with food and water ad libitum.
Rabbits were anesthetized with a combination of ketamine hydrochloride (35 mg/kg i.m.) and xylazine (5 mg/kg i.m.) and killed with an i.v. overdose of Valabarb (Jurox, Sydney, Australia). When required, the reproductive system of anesthetized rabbits was cleared of blood by perfusing saline through the vascular system via a cannula in the thoracic aorta.
Collection of Epididymal Fluid
Regions of the rabbit epididymis were identified according to the classification of Jones et al. [5]. Luminal fluid was sampled from region 8 of the epididymis by retrograde perfusion [8]. Samples from regions 2–5 were collected from epididymides that had been cleared of blood by perfusing the vascular system. The duct was then pierced with a sterile scalpel, and the contents were aspired into isotonic sperm buffer (ISB; 103 mM NaCl, 5 mM KCl, 3 mM MgCl2, 400 mM EDTA, 30 mM Tris-HCl, pH 7.2). Immediately after collection, samples were centrifuged (400 x g, 5 min) to separate sperm from the supernatant.
Isolation of REP38 and Preparation of Polyclonal Antibodies
REP38 was purified by preparative SDS-PAGE and electroelution from epididymal fluid collected from region 8 of the epididymis. Proteins were denatured and separated on a large format (16 x 18 cm) gel system (SE600; Hoefer Scientific Instruments, San Francisco, CA) with a 12% polyacrylamide resolving gel. Gels were loaded with 1 mg protein and electrophorosed at 100 V/gel. The protein bands identified as REP38 in six gels were pooled, homogenized in PBS (137 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.5 mM KCl, pH 7.4), electroeluted from the gel matrix within a dialysis membrane (molecular mass cutoff: 10 kDa), and collected following centrifugation. Protein purity was assessed on silver-stained [9] minigels, and the preparations were used to generate antibodies in two female Balb-C mice. The primary immunization included Freund complete adjuvant (40 µg REP38, i.p.), and two booster immunizations (20 µg REP38) with Freund incomplete adjuvant were administered at 2-wk intervals. Sera were prepared 10 days after the final booster and pooled following characterization by Western analysis.
Analysis of Anti-REP38 IgG Antibodies
Blood serum was purified in a Hi-Trap Protein G sepharose column (Pharmacia Biotech, Uppsala, Sweden), and the specificity of the purified IgG (anti-REP38 IgG) was determined by immunoblotting. Proteins present in fluid from region 8 of the rabbit epididymis were solubilized in either reducing buffer (130 mM Tris-HCl, pH 6.8, 20% v/v glycerol, 4% w/v SDS, 0.005% w/v bromophenol blue, 5% v/v ß-mercaptoethanol) or nonreducing buffer (130 mM Tris-HCl, pH 6.8, 20% v/v glycerol, 4% w/v SDS, 0.005% w/v bromophenol blue) and separated by SDS-PAGE. The proteins were also separated by two-dimensional electrophoresis. The isoelectric focusing was performed on immobilized pH gradient acrylamide gels (Immobiline DryStrips, 110 x 3 mm, pH 3–10; Pharmacia Biotech) using the following protocol: 300 V for 5 h; 1000 V for 10 h; 3500 V for 35 h. The second dimension separation was performed on large format 12% polyacrylamide gels (100 V/gel). Following electrophoresis, proteins were electrotransferred onto Polyscreen polyvinylidene fluoride (PVDF) transfer membranes (250 mA for 1 h) using a Mini Trans-Blot Cell (Bio-Rad, Richmond, CA).
Following transfer, the membranes were blocked in PBS with 5% skim milk (overnight at 4°C). Blots were then incubated in the presence of preimmune or immune IgG diluted (1 µg/50 ml) in PBS with 1% skim milk (1 h at room temperature). The blots were washed 3 times for 10 min each in PBS with 0.05% Tween-20 and incubated (1 h at room temperature) with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody diluted 1:2000. The membranes were again washed 3 times, and reactive bands were visualized by incubation in a solution of PBS with 0.5 mg/ml diaminobenzidine and 0.02% H2O2.
Immunohistochemistry
Tissue Tissues were fixed by perfusing the vascular system with saline and then Bouin fluid, then by immersion in Bouin fluid. Samples of the testis, epididymis, vas deferens, seminal vesicles, prostate, kidney, liver, and muscle were dehydrated in ethanol, embedded in paraffin, sectioned at 5 µm, mounted on poly-L-lysine-coated slides, deparaffinized in xylene, and rehydrated into PBS. Nonspecific antibody binding was blocked by overnight incubation in 3% bovine serum albumin (BSA) and PBS (4°C), and sections were incubated (1 h at room temperature) in a humidified chamber with the anti-REP38 IgG diluted (1 µg IgG/200 µl) in PBS with 1% BSA and then washed 3 times in PBS. Incubation with the secondary antibody was carried out using fluorescein isothiocynate (FITC)-labeled sheep anti-mouse IgG diluted 1:60 in PBS with 1% BSA. Slides were mounted with antifade reagent (SlowFade; Molecular Probes, Eugene, OR) and viewed with a confocal microscope (MRC-1000 laser scanning confocal imaging system; Bio-Rad). Sections were incubated in the presence of preimmune IgG or secondary antibody in all experiments as negative controls.
Sperm
Following separation from epididymal fluid, sperm were diluted 1000-fold in ISB and washed 3 times by gentle centrifugation (400 x g, 5 min). Washed spermatozoa (
5 x 104) were air-dried onto poly-L-lysine-coated glass microscope slides and fixed by immersion in methanol (10 min). Slides were then rinsed with ISB, immunostained with anti-REP38 IgG and FITC-labeled secondary antibody diluted 1:60 in ISB with 1% BSA as outlined for tissue sections, mounted with antifade, and viewed under the confocal microscope.
Alternatively, washed suspensions of live spermatozoa were diluted to a final concentration of 106 spermatozoa/ml in ISB with 3% (w/v) BSA and blocked for 1 h at 33°C. Blocking solution was removed by centrifugation (400 x g, 4 min), and sperm were resuspended directly in ISB containing 1% w/v BSA and immune IgG and incubated for 1 h at 33°C. The suspension was again washed by centrifugation (400 x g, 4 min), and the pellet was resuspended in ISB containing 1% (w/v) BSA and FITC-labeled secondary antibody and incubated in the dark for 1 h at 33°C. The suspension was washed as above, and the sperm pellet was resuspended for a final time in ISB. Sperm were then mounted onto poly-L-lysine-coated glass microscope slides and viewed immediately. The integrity of the sperm plasmalemma was monitored with the use of a monoclonal antibody (IT2A3) directed against a protein located on the internal acrosomal membranes (unpublished data). Intact sperm do not stain with this antibody.
Control incubations of live and methanol-fixed sperm were conducted by substituting immune IgG for preimmune IgG or by omitting the secondary antibody. These preparations showed no fluorescence above a residual background staining on any sperm.
Tissue Homogenates for Western Blots
Tissues cleared of blood were homogenized in buffer (0.25 M sucrose, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.4) using a polytron (2 min at 4°C). Homogenates were centrifuged (100 000 x g, 30 min, 2°C), and the supernatant, interpreted as containing soluble tissue proteins, was carefully removed and dialyzed against 3 changes of distilled water (24 h at 4°C). Protein concentrations were determined using the Protein Assay kit (Pierce, Rockford, IL).
Extraction of Sperm Proteins
Proteins were extracted from sperm by one of two methods. One method was an adaptation by M.K. Holland and J. Andrews (unpublished data) of a method for removing the sperm plasma membrane [10]. Sperm were washed 3 times using a 100-fold dilution in 50 mM Tris-HCl (pH 7.4) and repeated centrifugation (800 x g, 15 min, room temperature). The supernatants from each wash were collected, pooled, and dialyzed for 24 h at 4°C against 3 changes of distilled water. The other method involved a modified [11] sequential extraction protocol [12]. Sperm were sequentially extracted in ISB, high ionic strength PBS (PBS supplemented with 0.5 M NaCl, 5 mM EDTA), mild detergent (0.1% Triton X-100), and strong detergent (2% SDS) using a dilution rate of 1:100.
In Vitro Fertilization Assay
Rabbit oocytes were recovered from the oviducts of superovulated does [13], and capacitated sperm were collected from the uterine horns of a mature doe mated with a buck of proven fertility. The capacitated sperm were incubated (30 min at 37°C) with either preimmune IgG (400 µg/ml, control) or anti-REP38 IgG (40 or 400 µg/ml) in Brackett defined medium (BDM; 112 mM NaCl, 4.02 mM KCl, 2.25 mM CaCl2, 0.83 mM NaH2PO4, 0.052 mM MgCl2, 37 mM NaHCO3, 13.9 mM glucose, 1.25 mM sodium pyruvate, 3 mg/ml BSA, 0.031 mg/ml sodium penicillin) before being washed with 3 changes of fresh BDM to remove unbound antibody. Washed sperm (5 x 10 4) were added to cumulus-intact oocytes and incubated under oil in 5% CO2 (6 h at 37°C). Following incubation, oocytes were transferred to fresh BDM and incubated for a further 18 h at 37°C. Oocytes were then washed by gentle pipetting through a fine-bore pipette in 3 changes of BDM to remove loosely bound sperm. The number of sperm bound to the zona pellucida and present in the perivitelline space was recorded. Fertilization, defined as the presence of male and female pronuclei or cleavage of the embryo, was scored using Nomarski optics on a Zeiss compound microscope (Carl Zeiss, Thornwood, NY).
Statistics
Values are presented as mean ± SEM; the SEM was calculated from the variance between replicates of the experiment. Significance of differences was determined using ANOVAs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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REP38 was purified from luminal fluid from region 8 of the epididymis (Fig. 1, lane 1) to apparent homogeneity (Fig. 1, lane 2) by 2 successive rounds of preparative SDS-PAGE. Yields of purified protein from 1 mg of starting material were 25–50 µg, indicating that REP38 constitutes a minimum of 2.5%–5% of the protein in cauda epididymal fluid.
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Analysis of REP38 Polyclonal Antibody
Anti-REP38 immune IgG reacted with a predominant band in epididymal fluid of approximately 38 kDa corresponding to REP38 (Fig. 1, lanes 3 and 4). A weak cross-reaction was also detected with a second moiety of approximately 77 kDa (REP77). Both bands were recognized by anti-REP38 IgG under reducing and nonreducing conditions (Fig. 1, lanes 3 and 4, respectively), thereby indicating that REP77 is not a dimer of 38-kDa subunits joined by disulfide bonds. Blots of a two-dimensional gel of luminal fluid from region 8 showed that both proteins consist of several basic isoelectric charge variants with isoelectric points of approximately 6.2–6.9 (REP77) and 7.8–9.0 (REP38). Blots from which the primary antibody was omitted or replaced with preimmune IgG showed no cross-reactivity.
Tissue Specificity of REP38 Synthesis
Immunoblots of tissue extracts prepared from the epididymis, testis, ovary, brain, kidney, liver, spleen, and muscle showed that REP38 was exclusively detected in the epididymis (data not shown). Under both reducing and nonreducing conditions, two bands corresponding to REP38 and REP77 were observed in the epididymal extracts. No reactivity was seen in blots when primary antibody was either omitted or replaced with preimmune IgG.
Histological sections showed reactivity with anti-REP38 IgG for samples from the epididymis and vas deferens but not for samples from the testis, prostate, or seminal vesicles (Fig. 2). Only moderate immunoreactivity was detected in the vas deferens, and the staining was restricted to the lumen and the luminal border of the epithelium (Fig. 2B). No reactivity was seen when primary antibody was either omitted or replaced with preimmune IgG.
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Regional Synthesis of REP38 in the Epididymis
Region 5 was the most proximal region of the epididymis to stain when REP38 was localized immunocytochemically using HRP and FITC-labeled secondary antibodies (only the latter shown in Fig. 3). At the beginning of this region, the staining was restricted to the supranuclear cytoplasm of a limited number of principal cells, but staining increased in a short distance to the supranuclear region and the microvilli of all principal cells. Staining intensity increased through region 6, but in region 7 staining was less intense in the cytoplasm and was concentrated around the adluminal border and microvilli. There was heavy staining of the adluminal border and microvilli of principal cells in region 8, but there was little staining of the cytoplasm except in a few sporadically arranged cells in which the entire cytoplasm was stained.
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No staining was observed in basal cells or in peritubular connective tissue or vascular tissue. However, reaction product accumulated in the lumen at sites distal to its origin and was associated with spermatozoa. Some sperm were stained in region 5 of the epididymis, all appeared to be stained by region 6, and the staining intensity increased caudally. In no region of the epididymis was there any staining of control sections incubated in the presence of preimmune IgG or in the absence of either primary or secondary antibody.
Association of REP38 with Spermatozoa
Reactivity to anti-REP38 IgG was detected in <10% of sperm collected from regions 2–5 of the epididymis but in >90% of sperm from region 8 (Fig. 4). Labeling was identified in three discrete membrane domains of sperm: the acrosomal and postacrosomal regions and the middle piece. The patterns of localization were similar in live sperm and methanol-fixed preparations, suggesting that the antigens were predominantly associated with the sperm surface, and there were no substantial pools of antigen in internal membranes. The pattern was not altered during epididymal transit, but the relative staining intensity increased distally. This apparent increase was also detected on immunoblots of extracts of sperm membrane proteins (Fig. 5) and was thought to reflect an increase in the number of sperm labeled and an accumulation of REP38 bound to the surface of sperm.
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Proteins on sperm from region 8 of the epididymis were sequentially extracted to investigate the physical nature of the interaction between REP38 and the sperm surface. The predominant band of 38 kDa recognized by anti-REP38 IgG was partially extracted by isotonic solutions, more by high-salt buffer, and more by treatment with Triton X-100 (0.1%) (Fig. 6). Further treatment with SDS (2%) removed a detectable amount of the remaining antigen. Although some antigen may coat the sperm surface, a substantial amount binds very tightly, like an integral membrane protein, and is only removed by solubilization of the sperm membrane. By contrast, REP77 was completely extracted from sperm by the high-salt buffer (Fig. 6), and subsequent immunofluorescent labeling with anti-REP38 IgG was restricted to the middle piece and postacrosomal domains. Labeling was weak or absent in the acrosomal region (data not shown). This finding raises the possibility that REP38 and REP77 may exist as separate pools on the sperm surface.
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Immunofluorescent staining was performed on sperm exposed to capacitation conditions in the uterus of the doe and recovered by flushing 12 h postcoitum. The conditions considerably reduced labeling intensity and caused an apparent redistribution of the antigen on approximately 40% of spermatozoa, from their original positions in the acrosome and postacrosomal regions to the equatorial segment (Fig. 7). However, immunofluorescence persisted in the middle piece of these sperm.
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Live sperm were studied because of concerns that immunofluorescent techniques cannot distinguish between internal and surface antigens [14] and because the plasmalemma may be disrupted by treatments such as centrifugation [15]. A monoclonal antibody (IT2A3) specific to internal membrane proteins was used to show that the majority of sperm retained the integrity of the plasmalemma, with <5% displaying positive fluorescence for the intracellular marker (results not shown). In contrast, >90% of sperm fixed with methanol stained positive with this antibody.
In Vitro Fertilization
Table 1 shows that preincubation of sperm with anti-REP38 IgG had a significant (P < 0.05) concentration-dependent inhibitory effect on in vitro fertilization. At concentrations of 40 µg/ml and 400 µg/ml, the immune IgG reduced the fertilization rate to around 25% and 6%, respectively, of the samples preincubated with preimmune serum (i.e., 75% and 94% inhibition, respectively). Sperm remained freely motile after incubation in anti-REP38 IgG, suggesting that the inhibitory effect was not associated with a reduction in sperm motility or sperm agglutination. About the same number of sperm were bound to the zona pellucida as were present within the perivitelline space of eggs in all treatment groups, including the control group treated with preimmune serum. Consequently, the reduction in fertilization rate was considered related to interference with fusion between sperm and egg membranes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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REP38 was localized immunocytochemicaly within the supranuclear region of principal cells of the epididymal epithelium. This finding is consistent with it being synthesized and delivered as a merocrine secretion [5, 20]. The transition from this pattern of localization in all principal cells to occurrence in the supranuclear cytoplasm of a few scattered principal cells and the microvillar surface in region 8 is similar to the pattern reported for rabbit uteroglobin [17]. The occurrence in epithelium lining region 8 raises the possibility that there may be some reabsorption of the protein in this region. This possibility is consistent with the ultrastructure of the epithelium and the demonstration of selective absorption of labeled compounds in region 8 of the rabbit epididymis [5, 21]. Nevertheless, it does not reflect what is happening to other proteins; there is net protein secretion between regions 5 and 8 [4].
Results from immunolocalization and immunoblotting confirmed that REP38 associates with spermatozoa during their passage through the epididymis [4]. REP38 first associated with spermatozoa within region 5 of the epididymis, and both the intensity of fluorescence and the proportion of sperm displaying positive fluorescence increased distally. The increase was confirmed by Western blot analysis. This finding is consistent with findings for other proteins [16, 22–24] and implies that there is a progressive association of rabbit epididymal proteins with the sperm surface. It is unknown whether this phenomenon is facilitated by biochemical changes of the sperm surface or is the result of a time-dependent binding of the proteins. The variations in staining may also reflect the increase in luminal concentration of REP38 from region 5 to region 8 of the epididymis.
Anti-REP38 IgG was localized to three sperm domains: the acrosomal and postacrosomal regions of the head and the middle piece. These domains are identical to those identified by Garcia et al. [18] using an antibody against total rabbit epididymal proteins, indicating that rabbit epididymal proteins have an affinity for specific domains on the surface of maturing sperm. This pattern of association occurs in a number of species for several sperm-associated epididymal proteins [3, 15–17, 22–25]; however, the mechanism of the association has not been established. One possibility is that an interaction is mediated through high-affinity protein binding sites on the sperm surface, as proposed for the domain-specific targeting of the rat epididymal protein DE [26]. This possibility is consistent with the finding that destabilization of the sperm membrane with detergent was necessary to completely extract REP38.
The occurrence of REP38 on the sperm surface does not change as sperm pass through the epididymis, consistent with reports that the plasmalemma of rabbit sperm is stabilized in the epididymis [23, 25] by acrosome stabilizing factor (ASF [18, 23, 25, 27, 28]), a heterodimeric glycoprotein. Coincident with the loss of ASF under in vivo capacitating conditions [29], REP38 redistributed to a new domain, the equatorial segment of the acrosome. The failure to induce this change in localization by permeabilization of the sperm plasmalemma or by treatment in high ionic strength medium argues that the redistribution is the result of protein migration rather than exposure of a second population of REP38 previously cryptic on intact sperm. The migration of REP38 under capacitating conditions is consistent with the finding that sperm surface antigens migrate during capacitation and the acrosome reaction in a number of other species [30–34] and also with findings of an earlier report that the plasmalemma of rabbit sperm is remodeled after ejaculation [35].
The selective redistribution of REP38 from a plasma membrane domain that is lost during the acrosome reaction [36] indicates that the protein is not involved in the interaction between sperm and the zona pellucida. However, REP38 moves to the equatorial segment of the acrosome, indicating that it may be involved in membrane fusion between sperm and ovum [37]. This proposal is consistent with the findings of the in vitro fertilization assay, which showed that anti-REP38 IgG significantly reduced the fertilizing ability of mature spermatozoa. The data also indicate that the immune inhibition was effective after sperm-zona binding; about the same number of sperm bound to the zona as were present in the perivitilline space of sperm incubated in anti-REP IgG and preimmune IgG. This finding precludes the possibility that the inhibition was an artifact associated with steric hindrance and indicates an interference of the ability of sperm to interact with the oolemma.
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FOOTNOTES |
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1 This work was supported by a grant from the Australian Research Council, the Research Management Committee, University of Newcastle, and the Vertebrate Biocontrol Cooperative Research Centre, Canberra, Australia. B.N. was supported by an Australian Postgraduate Award and a Pest Animal Control Cooperative Research Centre Student Award. 
2 Correspondence: Michael Holland, Pest Animal Control Cooperative Research Centre, CSIRO Sustainable Ecosystems, P.O. Box 284, Canberra, ACT 2601, Australia. FAX: 61 2 6242 1511; michael.holland{at}csiro.au
Accepted: February 22, 2002.
Received: April 8, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. Nixon, C. M. Hardy, R. C. Jones, J. B. Andrews, and M. K. Holland Rabbit Epididymal Secretory Proteins. III. Molecular Cloning and Characterization of the Complementary DNA for REP38 Biol Reprod, July 1, 2002; 67(1): 147 - 153. [Abstract] [Full Text] [PDF]
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