Rabbit Epididymal Secretory Proteins. II. Immunolocalization and ...

BIOLOGY OF REPRODUCTION 67, 140–146 (2002)

Rabbit Epididymal Secretory Proteins. II. Immunolocalization and Sperm Association of REP381 Brett Nixon,3,4 Russell C. Jones,4 Hannah G. Clarke,3 and Michael K. Holland2,3 Pest Animal Control Cooperative Research Centre,3 CSIRO Sustainable Ecosystems, Canberra, ACT 2601, Australia Department of Biological Sciences,4 University of Newcastle, Callaghan, NSW 2308, Australia ABSTRACT

MATERIALS AND METHODS

Polyclonal antibody was used to partially characterize REP38, a major rabbit epididymal secretory protein. Western blot analyses and immunohistochemistry indicated that REP38 is only expressed in regions 5 and 6 of the epididymis (corpus epididymidis) and is localized in the supranuclear region and microvilli of the principal cells in these regions. It was not expressed in other tissues of the body. In region 8 (cauda epididymidis), REP38 was detected in the luminal border and cytoplasm of scattered principal cells, indicating that it may be reabsorbed in this region. This protein accumulated on the sperm plasma membrane downstream of region 5 and was localized predominantly over the acrosomal and postacrosomal regions of the head and the middle piece. Although tightly bound to epididymal sperm, REP38 migrated to the equatorial segment under conditions in vivo that would promote capacitation. When tested in vitro, anti-REP38 IgG reduced the percentage of ova fertilized in a concentration-dependent manner, apparently by blocking sperm-egg fusion.

Animals 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 3 g, 5 min) to separate sperm from the supernatant.

epididymis, gamete biology, sperm maturation

INTRODUCTION

Isolation of REP38 and Preparation of Polyclonal Antibodies

The mammalian epididymis plays an important role in reproduction by maturing and storing sperm [1]. The maturation process is dependent on sperm associating with specific proteins secreted by the epididymal mucosa under the influence of androgen [2, 3]. In an earlier report on the rabbit [4], we identified a number of proteins secreted by the epididymal mucosa and their dependence on androgen. Here, we describe the isolation, characterization and immunolocalization of the most abundant of the secreted proteins that is present in luminal fluid from the cauda epididymidis, REP38, a protein of Mr 38 000. This protein was present in luminal fluid flushed from epididymal regions 2– 5, 7, and 8 (classification according to Jones et al. [5]), its secretion is androgen dependent, and it associates with sperm during passage through the epididymis. REP38 is homolgous with two rat testicular cDNAs, Odf2 and KTT4, which putatively encode outer dense fiber proteins [6, 7].

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 3 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 mg REP38, i.p.), and two booster immunizations (20 mg 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 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; e-mail: michael.holland@csiro.au 1

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 b-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 3 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)

Received: 8 April 1999. First decision: 24 May 1999. Accepted: 22 February 2002. Q 2002 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

140

IMMUNOLOCALIZATION OF REP38

141

transfer membranes (250 mA for 1 h) using a Mini Trans-Blot Cell (BioRad, Richmond, CA). Following transfer, the membranes were blocked in PBS with 5% skim milk (overnight at 48C). Blots were then incubated in the presence of preimmune or immune IgG diluted (1 mg/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 mm, 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 (48C), and sections were incubated (1 h at room temperature) in a humidified chamber with the anti-REP38 IgG diluted (1 mg IgG/200 ml) 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 3 g, 5 min). Washed spermatozoa (;5 3 104) were air-dried onto poly-Llysine-coated glass microscope slides and fixed by immersion in methanol (10 min). Slides were then rinsed with ISB, immunostained with antiREP38 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 338C. Blocking solution was removed by centrifugation (400 3 g, 4 min), and sperm were resuspended directly in ISB containing 1% w/v BSA and immune IgG and incubated for 1 h at 338C. The suspension was again washed by centrifugation (400 3 g, 4 min), and the pellet was resuspended in ISB containing 1% (w/v) BSA and FITClabeled secondary antibody and incubated in the dark for 1 h at 338C. 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-lysinecoated 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 48C). Homogenates were centrifuged (100 000 3 g, 30 min, 28C), and the supernatant, interpreted as containing soluble tissue proteins, was carefully removed and dialyzed against 3 changes of distilled water (24 h at 48C). 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 3 g, 15 min, room temperature). The supernatants from each wash were collected, pooled, and dialyzed for 24

FIG. 1. Partial purification of REP38 from luminal fluid from region 8 of the epididymis, and assessment of anti-REP38 IgG specificity by Western blotting. Lane 1: region 8 fluid and identification of REP38; lane 2: REP38 purified by preparative electrophoresis and electroelution. This fraction was used for the subsequent generation of murine polyclonal antibodies; lanes 3 and 4: immunoblots of region 8 epididymal fluid resolved under reducing and nonreducing conditions, respectively. Blots were immunostained with anti-REP38 IgG. Numerical values on the left correspond to the molecular weight of the two immunoreactive bands.

h at 48C 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 378C) with either preimmune IgG (400 mg/ml, control) or anti-REP38 IgG (40 or 400 mg/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 3 10 4) were added to cumulus-intact oocytes and incubated under oil in 5% CO2 (6 h at 378C). Following incubation, oocytes were transferred to fresh BDM and incubated for a further 18 h at 378C. Oocytes were then washed by gentle pipetting through a finebore 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 6 SEM; the SEM was calculated from the variance between replicates of the experiment. Significance of differences was determined using ANOVAs.

142

NIXON ET AL.

FIG. 2. Immunofluorescent localization of REP38 in the male rabbit gential tract. Composite phase contrast (left side of each panel) and immunofluorescence (right side of each panel) photomicrographs demonstrating the localization of anti-REP38 IgG on histologic sections of the testis (A), vas deferens (B), seminal vesicles (C), and prostate (D). Immunoreactivity was detected in the lumen of the vas deferens. All other tissues stained negative. e, Epithelium; l, lumen. Bars 5 50 mm.

RESULTS

Tissue Specificity of REP38 Synthesis

Purification of REP38

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.

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 SDSPAGE. Yields of purified protein from 1 mg of starting material were 25–50 mg, indicating that REP38 constitutes a minimum of 2.5%–5% of the protein in cauda epididymal fluid. 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 crossreaction 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.

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

IMMUNOLOCALIZATION OF REP38

143

FIG. 3. Immunofluorescent localization of REP38 in the epididymis. A) Region 5 of the epididymis was the most proximal site showing immunoreactivity. Staining was restricted to the supranuclear region (SN) and microvilli (MV) of principal cells in regions 5 and 6 of the epididymis. Sperm (SP) within the lumen (L) are also stained at sites adjacent to REP38 synthesis. B) Staining intensity increased through region 6, where virtually all principal cells were uniformly stained. C) In region 7, cytoplasmic staining of epithelial cells diminished. However, intense staining of the microvillar border was apparent. D) Staining was present in the cytoplasm of isolated cells in region 8.

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. 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. 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

144

NIXON ET AL.

FIG. 4. Immunofluorescent localization of REP38 on spermatozoa. Control sperm (A) were incubated with preimmune IgG. Live sperm from region 8 (B) were stained with anti-REP38 antibody. Methanol-fixed sperm from region 5 (C) and region 8 (D) stained with anti-REP38 antibody. Fluorescence was restricted to the anterior and posterior acrosomal regions of the head and to the middle piece of the tail of both live (B) and methanol-fixed (C and D) spermatozoa.

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. 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. 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 centrifu-

FIG. 5. Extraction of sperm plasma membrane proteins. Proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-REP38 antibodies. Lanes contain epididymal fluid (F) and extracts of sperm plasma membranes (PM) from regions 2–5 and 8 of the epididymis.

gation [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 antiREP38 IgG had a significant (P , 0.05) concentration-dependent inhibitory effect on in vitro fertilization. At concentrations of 40 mg/ml and 400 mg/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

FIG. 6. Sequential extraction of sperm proteins. Proteins from sperm from region 8 of the epididymis were extracted, resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-REP38 antibodies. Lanes are epididymal fluid from region 8 (F); proteins sequentially extracted with low-salt buffer (LS), high-salt buffer (HS), Triton X-100 (T), and SDS (SDS); and proteins extracted sequentially with isotonic buffer (IS1–IS3).

145

IMMUNOLOCALIZATION OF REP38 TABLE 1. Effect of anti-REP38 IgG on fertilization rates of rabbit oocytes in vitro. Antibody treatmenta REP38 Variable Replicatec Total eggs Zona-bound sperm (mean 6 SEM) Sperm in perivitelline (mean 6 SEM) % Eggs fertilized (mean 6 SEM)

Controlb

40 mg/ml

400 mg/ml

3 24 5.2 6 2.9 0.7 6 0.5 71 6 2.4

3 33 3.3 6 2.3 0.5 6 0.4 18 6 0.4d

3 21 2.5 6 2.1 0.3 6 0.2 4.7 6 0.5d

a Capacitated sperm (from three donors of proven fertility) were diluted to a concentration of 5 3 105 in 1 ml BDM and incubated with anti-REP38 IgG (40 or 400 mg IgG/ml) for 30 min at 378C. A 100-ml aliquot of this suspension (;5 3 104 sperm) was added to the oocytes. Oocytes were then incubated for 24 h before scoring for the number of sperm tightly bound, sperm penetrated to the perivitelline space, and oocytes fertilized. b In vitro control sperm were incubated with 400 mg IgG/ml preimmune IgG. c Seven to 11 eggs (from four donor females) were used in each treatment group in each replicate of the experiment. SEMs were calculated from the variance between replicates. d P , 0.05, comparison by one-way ANOVA with in vitro controls.

with preimmune serum. Consequently, the reduction in fertilization rate was considered related to interference with fusion between sperm and egg membranes. DISCUSSION

The results described here confirm our previous proposal [4] that REP38 is a protein that is synthesized and secreted by the epididymis. Use of a polyclonal antibody confirmed that REP38 is not present in tissues outside the epididymis and clarified that REP38 is only synthesized and secreted in regions 5 and 6 of the epididymis and persists caudally in the lumen. The regional synthesis of REP38 along the epididymis resembles that of several rabbit epididymal proteins [16–19] and may reflect localized specific functions of these proteins. 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 timedependent 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

FIG. 7. Immunofluorescent localization of REP38 on capacitated spermatozoa. Capacitated sperm recovered from uterine flushings of mated female rabbits (12 h postcoitum) were stained sequentially with antiREP38 IgG and FITC-conjugated anti-mouse IgG. Bar 5 10 mm.

146

NIXON ET AL.

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 antiREP 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.

16. 17. 18.

19. 20. 21. 22.

23.

24.

REFERENCES 1. Jones RC. Evolution of the vertebrate epididymis. J Reprod Fertil 1998; 53(suppl):163–181. 2. Orgebin-Crist MC, Jahad N. The maturation of rabbit epididymal spermatozoa in organ culture: stimulation by epididymal cytoplasmic extracts. Biol Reprod 1979; 21:511–515. 3. Jones R. Plasma membrane structure and remodelling during sperm maturation in the epididymis. J Reprod Fertil 1998; 53(suppl):73–84. 4. Nixon B, Jones RC, Hansen LA, Holland MK. Rabbit epididymal secretory proteins. I. Characterization and hormonal regulation. Biol Reprod 2002; 67:133–139. 5. Jones R, Hamilton DW, Fawcett DW. Morphology of the epithelium of the extratesticular rete testis, ductuli efferentes and ductus epididymidis of the adult male rabbit. Am J Anat 1979; 156:373–400. 6. Brohmann H, Pinnecke S, Hoyer-Fender S. Identification and characterization of new cDNAs encoding outer dense fibre proteins of rat sperm. J Biol Chem 1997; 272:10327–10332. 7. Turner KJ, Sharpe RM, Gaughan J, Millar MR, Foster PM, Saunders PT. Expression cloning of a rat testicular transcript abundant in germ cells, which contains two leucine zipper motifs. Biol Reprod 1997; 57:1223–1232. 8. Sujarit S, Jones RC, Setchell BP, Chaturapanich G, Lin M, Clulow J. Stimulation of protein secretion in the initial segment of the rat epididymis by fluid from the ram rete testis. J Reprod Fertil 1990; 88: 315–321. 9. Morrisey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117:307–310. 10. Fayrer-Hosken RA, Caudle AB, Shur BD. Galactosyltransferase activity is restricted to the plasma membranes of equine and bovine sperm. Mol Reprod Dev 1991; 28:74–78. 11. Murphy GJP, Carroll J. Detection and localization of antigens on the surface of mouse spermatozoa. Biochem J 1987; 241:379–387. 12. Rankin TL, Tsuruta KJ, Holland MK, Griswold MD, Orgebin-Crist MC. Isolation, immunolocalization, and sperm-association of three proteins of 18, 25, and 29 kilodaltons secreted by the mouse epididymis. Biol Reprod 1992; 46:747–766. 13. Brackett BG, Oliphant G. Capacitation of rabbit sperm in vitro. Biol Reprod 1975; 12:260–274. 14. Jones R, von Glos KI, Brown CR. Changes in the protein composition of rat spermatozoa during maturation in the epididymis. J Reprod Fertil 1983; 67:299–306. 15. Phillips DM, Jones R, Shalgi R. Alterations in distribution of surface

25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35.

36. 37.

and intracellular antigens during epididymal maturation of rat spermatozoa. Mol Reprod Dev 1991; 29:347–356. Moore HDM. Localization of specific glycoproteins secreted by the rabbit and hamster epididymis. Biol Reprod 1980; 22:705–718. El Etreby MF, Beier HM, Elger W, Mahrouse AT, Topert M. Immunocytochemical localization of uteroglobin in the genital tract of male rabbits. Cell Tissue Res 1983; 229:61–73. Garcia C, Regalado F, Lo´pez de Haro MS, Nieto A. Ultrastructural localization of epididymal secretory proteins associated with the surface of spermatozoa from rabbit cauda epididymis. Histochem J 1988; 20:708–714. Reynolds AB, Thomas TS, Wilson WL, Oliphant G. Concentration of acrosome stabilising factor (ASF) in rabbit epididymal fluid and species specificity of anti-ASF antibodies. Biol Reprod 1989; 40:673–680. Nicander L, Malmqvist M. Ultrastructural observations suggesting merocrine secretion in the initial segment of the mammalian epididymis. Cell Tissue Res 1977; 184:487–490. Nicander L, Ploen L. Studies on regional fine structure and function in the rabbit epididymis. Int J Androl 1979; 2:463–481. Moore HDM. Glycoprotein secretions of the epididymis in the rabbit and hamster: localization on epididymal spermatozoa and the effect of specific antibodies on fertilization in vivo. J Exp Zool 1981; 215: 77–85. Thomas TS, Reynolds AB, Oliphant G. Evaluation of the site of synthesis of rabbit sperm acrosome stabilizing factor using immunocytochemical and metabollic labeling techniques. Biol Reprod 1984; 30: 693–705. Nieto A, Gutierrez Sagal R, Perez Martinez M. Isolation and characterization of a rabbit epididymal secretory glycoprotein that associates to the spermatozoon surface. Mol Reprod Dev 1997; 46:337– 343. Thomas TS, Reynolds AB, Wilson WL, Oliphant G. Chemical and physical characterisation of rabbit acrosome stabilising factor. Biol Reprod 1986; 35:691–703. Hall JC, Tubbs CE, Li Yuu, Ashraf S. Characterization of high-affinity protein D binding sites on the surface of rat epididymal spermatozoa. Biochem Mol Biol Int 1996; 40:1003–1010. Eng LA, Oliphant G. Rabbit sperm reversible decapacitation by membrane stabilisation with a highly purified glycoprotein from seminal plasma. Biol Reprod 1978; 19:1083–1094. Wilson WL, Oliphant G. Isolation and biochemical characterisation of the subunits of the rabbit acrosome stabilising factor. Annu Rev Biochem 1987; 37:159–169. Oliphant G, Singhas CA. Iodination of rabbit sperm plasma membrane: relationship of specific surface proteins to epididymal function and sperm capacitation. Biol Reprod 1979; 21:937–944. Myles DG, Primakoff P. Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization. J Cell Biol 1984; 99:1631–1641. Saxena NK, Russell LD, Saxena N, Peterson RN. Immunofluorescence antigen localization on boar sperm plasma membranes: monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during sperm maturation and at ejaculation. Anat Rec 1986; 214:238–252. Lopez LC, Shur BD. Redistribution of mouse sperm surface galactosyltransferase after the acrosome reaction. J Cell Biol 1987; 105: 1663–1670. Jones R, Shalgi R, Hoyland J, Phillips DM. Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro. Dev Biol 1990; 139:349–362. Rochwerger L, Cuasnicu PS. Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation. Mol Reprod Dev 1992; 31:34–41. Nicolson GL, Usui N, Yanagimachi R, Yanagimachi H, Smith JR. Lectin-binding sites on the plasma membranes of rabbit spermatozoa. Changes in surface receptors during epididymal maturation and after ejaculation. J Cell Biol 1977; 74:950–962. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, vol. 1. New York: Raven Press; 1994: 189–317. Bedford JM, Moore HDM, Franklin LE. Significance of the equatorial segment of the acrosome of the spermatozoa in eutherian mammals. Exp Cell Res 1979; 119:119–126.