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a Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37205
ABSTRACT
Even though the epididymis produces an environment promoting sperm maturation and viability, some sperm do not survive transit through the epididymal tubule. Mechanisms that segregate the epididymal epithelium and/or the viable sperm population from degenerating spermatozoa are poorly understood. We report here the identification and characterization of HEP64, a 64-kDa glycoprotein secreted by principal cells of the corpus and proximal cauda epididymidis of the hamster that specifically binds to and coats dead/dying spermatozoa. The HEP64 monomer contains
12 kDa carbohydrate and, following chemical deglycosylation, migrates as a 52-kDa polypeptide. Both soluble (luminal fluid) and sperm-associated HEP64 are assembled into disulfide-linked high molecular weight oligomers that migrate as a doublet band of 260/280 kDa by nonreducing SDS-PAGE. In the epididymal lumen, HEP64 is concentrated into focal accumulations containing aggregates of structurally abnormal or degenerating spermatozoa, and examination of sperm suspensions reveals that HEP64 forms a shroudlike coating surrounding abnormal spermatozoa. The HEP64 glycoprotein firmly binds degenerating spermatozoa and is not released by either nonionic detergent or high salt extraction. Electron microscopic immunocytochemistry demonstrates that HEP64 localized to an amorphous coating surrounding the abnormal spermatozoa. The potential mechanisms by which this epididymal secretory protein binds dead spermatozoa as well as its possible functions in the sperm storage function of the cauda epididymidis are discussed.
epididymis, sperm, sperm maturation
INTRODUCTION
A pivotal function of the epididymis is the production of a luminal environment that promotes both the maturation and survival of spermatozoa [1, 2]. The epithelial principal cells that exhibit region-specific differences along the epididymal tubule in their structure and patterns of protein secretion regulate the luminal fluid composition [3]. The region-specific modification of the luminal environment is functionally related to the fact that sperm develop fertilizing capacity and forward motility at specific sites within the epididymis [4, 5]. While testosterone is required for epididymal function and post-testicular sperm maturation, the mechanisms regulating region-specific epithelial cell function are incompletely understood [6–8]. Testis-derived factors are critical for the functional differentiation of the initial segment, and luminal fluid constituents may also function in a paracrine manner to regulate epithelial cell function in successive epididymal segments [1, 9].
As sperm transit through the epididymis and interact with the luminal fluid, specific domains of their plasma membrane are remodeled by the binding of epididymal secretory proteins and by enzymatic processing [10, 11]. These surface changes appear critical to the development of fertilizing capacity, and in most species the maturation process is completed when spermatozoa reach the proximal cauda epididymidis, while the distal cauda region functions in sperm storage [4, 12]. The epididymal epithelium also secretes several proteins that promote sperm survival. These include enzymes involved in glutathione conjugation and metabolism [2, 6] that provide protection against oxidative damage, sulfated glycoprotein-2 (SGP-2 or clusterin) [13–15] that is proposed to inhibit complement-mediated cell lysis, and potential protease inhibitors such as HE4 [16] and CRES [17] that may protect spermatozoa from proteolytic degradation.
Although the epididymal environment actively promotes their maturation and survival, not all spermatozoa remain viable during passage through the epididymis. In the hamster the nonviable sperm population increases along the length of the epididymis [18]. In several species, aggregated masses of degenerating spermatozoa that are embedded in an amorphous electron-dense material have been noted within the lumen of the cauda epididymidis and vas deferens [19–21]. It is unclear whether this coating material represents sperm breakdown products or is derived from other sources. During our analysis of specific sperm fractions, we unexpectedly identified a minor glycoprotein of epididymal origin, with binding affinity for degenerating spermatozoa. This 64-kDa epididymal protein, termed HEP64, specifically coats the nonviable spermatozoa within the distal segments of the epididymis. In this study we report the identification, biochemical characterization, and regional expression of HEP64 in the hamster epididymis. These data suggest several possible roles for HEP64 in the sperm storage function of the epididymis.
MATERIALS AND METHODS
Preparation of Epididymal Sperm and Luminal Fluid Samples
Care and use of animals conformed to National Institutes of Health (NIH) guidelines for humane animal care and use in research, and all protocols were approved by the institutional animal care committee. Mature male golden hamsters were housed in the University animal care facility on a 14L:10D cycle. Animals were asphyxiated with CO2, and the caput and cauda epididymides were dissected and minced in calcium-free Tyrode solution at 37°C. The sperm suspension was centrifuged at 100 x g for 1 min to sediment tissue fragments, and the supernatant fluid was recentrifuged at 1500 x g for 10 min at 4°C. Sperm pellets were used in the extraction and fractionation protocols described below, while the supernatant fluids, representing crude caput and cauda epididymal luminal fluids, were centrifuged at 100 000 x g for 20 min in a Beckman TL55 rotor, dialyzed overnight at 4°C against 4 L of water containing 1 mM benzamidine, and lyophilized to powder.
Sperm Fractionation
An acrosomal matrix fraction was isolated from hamster cauda epididymal spermatozoa as described previously [22]. Briefly, spermatozoa were suspended into a Tris-saline-protease inhibitor solution (TNI = 150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 2 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.05% sodium azide) containing 0.1% Triton X-100 (TNI-Triton) at 4°C and then centrifuged at 1500 x g for 10 min; the sperm pellet was resuspended into TNI-Triton solution containing 0.5 M NaCl and extracted 1 h at 4°C. The sperm suspension was homogenized with a glass-Teflon homogenizer, mixed with five volumes of a solution containing 43% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ), 0.25 M sucrose, and 0.1% Triton X-100 in 50 mM Tris-HCl, pH 7.5, and centrifuged at 28 000 rpm for 35 min in a Beckman 60Ti rotor. The acrosomal matrices banded at the top of the gradient, and they were collected and pelleted by centrifugation at 35 000 rpm for 60 min in a Beckman SW41 rotor.
The acrosomal matrix fraction was further fractionated by high pH extraction with 100 mM CAPS (3-cyclohexamino-1-propanesulfonic acid; Sigma Chemical Co., St. Louis, MO), pH 10.5, containing 50 mM benzamidine for 2 h at 37°C [23]. The soluble and insoluble fractions were collected following centrifugation at 40 000 rpm for 30 min in a Beckman TL-55 rotor.
Antibody Preparation
Two cycles of SDS-PAGE [24] were utilized to purify HEP64 from the acrosomal matrix fraction. First, the high molecular weight, disulfide-linked, oligomeric form of HEP64 was isolated under nonreducing conditions by continuous elution SDS-PAGE (model 491 Prep Cell, Bio-Rad Laboratories, Hercules, CA) on a 4% acrylamide high porosity tube gel prepared with a 30:0.4 acrylamide:bisacrylamide ratio. The HEP64-containing fractions were then reduced in sample buffer containing 5% dithiothreitol (DTT) and fractionated by SDS-PAGE on a preparative 3–19% high porosity gel [24]. Polypeptides were transferred to nitrocellulose [25] and HEP64 was localized by staining separate lanes with [cf2]Ricinus communis[cf1] 120 agglutinin (RCA) and with colloidal gold [26]. The remainder of the HEP64 band was excised, solubilized with a small amount of dimethyl sulfoxide, and used to prepare monospecific antibodies in rabbits [27]. Animals were killed with sodium pentobarbital, and blood was collected by cardiac puncture for preparation of immune serum. Preimmune serum was prepared from blood collected prior to immunization.
Western Blotting
Polypeptides were separated by either SDS-PAGE [24] or two-dimensional PAGE [28] and electrophoretically transferred to polyvinylidene difluoride or nitrocellulose membranes [25]. High porosity SDS-PAGE was performed on 3–19% linear gradient separation gels prepared with a 30:0.4 acrylamide:bisacrylamide ratio, and routine SDS-PAGE was performed on 12% separation gel prepared with a 30:0.8 acrylamide:bisacrylamide ratio. Protein concentration of samples was estimated by the method of Bradford [29]. To visualize the total polypeptide pattern, lanes were stained with Coomassie blue [30].
Immunoblots were first blocked with PBS (150 mM NaCl, 20 mM sodium phosphate, pH 7.6) containing 0.1% Tween 20, 5% goat serum, 5% nonfat dry milk, and 2.5% BSA and then incubated with immune or preimmune serum diluted in PBS containing 0.1% Tween 20 and 1% goat serum (PBS-Tw-GS). After three washes in PBS-Tw-GS, the blots were incubated in an affinity-purified horseradish peroxidase-conjugated secondary antibody (KPL Inc., Gaithersburg, MD) diluted in PSB-Tw-GS; following several washes immunoreactive bands were visualized by color development with diaminobenzidine and H2O2.
Lectin-blot analysis was used to identify glycosylated polypeptides. Blots were blocked for 1 h in lectin buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 2 mM MnCl2, and 2 mM CaCl2) containing 1% BSA and 0.1% Tween 20 and then incubated 1 h in lectin buffer containing 0.5% hemoglobin and 5 µg/ml biotinylated RCA (Vector Laboratories, Burlingame, CA). After three rinses in lectin buffer, blots were incubated for 1 h in avidin-peroxidase complex (Biomedia Inc., Foster City, CA) and lectin-binding bands were visualized by color development as above.
The carbohydrate content of HEP64 was estimated by SDS-PAGE following chemical deglycosylation in trifluoromethanesulfonic acid for 1 h on dry ice [31].
Immunocytochemistry
Epididymides were immersed in OCT, frozen in liquid nitrogen, and 4- to 6-µm-thick cryosections were prepared and fixed 1 h with 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Sperm suspensions were fixed for 1 h at 4°C with 4% formaldehyde, 0.1 M sodium phosphate buffer, pH 7.4, and placed on poly-L-lysine-coated coverslips. Some sperm samples were permeabilized prior to fixation by incubation on ice for 30 min in TNI containing 0.25% Triton X-100; alternatively, following 30 min of formaldehyde fixation, sperm were then permeabilized by adding Triton X-100 to a final concentration of 0.25%.
Specimens were then rinsed in TNT (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.05% Tween 20), blocked in TNT containing 5% normal donkey serum and 2.5% BSA, and then incubated in immune serum diluted 1:500–1:2500 in blocking solution; parallel controls were incubated with an equal dilution of preimmune serum. The specimens were rinsed three times in TNT and incubated in an affinity-purified CY3-donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA). Following several rinses in TNT the samples were photographed using phase-contrast and epifluorescence microscopy.
Spermatozoa were prepared for immunoelectron microscopy using slight modifications of the above protocol. These included the substitution of equivalent levels of normal goat serum for donkey serum in all solutions and the use of 10-nm gold-conjugated goat anti-rabbit IgG as the secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ). Following the immunolabeling protocol, samples were fixed with 4% glutaraldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide, and embedded in Embed 812 [32]. Thin sections were stained with uranyl acetate and lead citrate.
RESULTS
Identification and Biochemical Characterization of HEP64
The HEP64 glycoprotein was initially identified in an acrosomal matrix fraction isolated from cauda epididymal spermatozoa. By SDS-PAGE the acrosomal matrix fraction contains major polypeptides of 29 kDa and 22 kDa, previously termed AM29 and AM22 [33], as well as several minor polypeptide bands (Fig. 1A, lane 1). Lectin staining identified a minor 64-kDa polypeptide (HEP64) in the acrosomal fraction that stained intensely with RCA, a lectin specific for oligosaccharides ending in galactose or N-acetylgalactosamine (Fig. 1B, lane 1); the major bands AM22 and AM29 did not bind RCA. The 64-kDa band also bound the lectins concanavalin A and wheat germ agglutinin (not shown). To define the solubility properties of HEP64, the acrosomal matrix fraction was subjected to high pH extraction with 100 mM CAPS (pH 10.5), and soluble and insoluble fractions were obtained by centrifugation. Coomassie blue staining revealed that most of the minor polypeptides remained associated with the insoluble fraction (Fig. 1A, lane 2), whereas AM29 and AM22 partitioned to the high-pH-soluble fraction (Fig. 1A, lane 3). The RCA-stained blots demonstrated that HEP64 partitioned primarily to the high-pH-insoluble fraction (Fig. 1B, lanes 2 and 3). These data demonstrate that the particulate sperm fraction contains a minor, high-pH-insoluble glycoprotein termed HEP64.
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To identify protein-protein interactions that could regulate HEP64 solubility, the acrosomal matrix fraction was subjected to nonreducing SDS-PAGE on high porosity 3–19% gradient gels. Coomassie blue staining demonstrated that unreduced AM22 and AM29 exhibited the same electrophoretic mobility as the reduced forms (Fig. 2, lane 1); in contrast, the major RCA-binding polypeptide migrated as a doublet band of 280 and 260 kDa, respectively, and no lectin-binding band at 64 kDa was noted (Fig. 2, lane 2). These data indicate that HEP64 is associated with a disulfide cross-linked polypeptide complex.
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Because native HEP64 is a disulfide-linked oligomer, two sequential cycles of SDS-PAGE, first under nonreducing and then under reducing conditions, were utilized to purify the RCA-binding HEP64 monomer from the high-pH-insoluble acrosomal fraction (Fig. 2, lanes 3 and 4). The 64-kDa band was excised and used to prepare a monospecific antiserum. Immunoblot analysis of the acrosomal fraction separated by nonreducing SDS-PAGE revealed that anti-HEP64 reacted only with a doublet band of 280 and 260 kDa (Fig. 2, lane 5), and no bands were noted with preimmune serum (Fig. 2, lane 6). This demonstrates that under nonreducing conditions no monomeric HEP64 is detectable. However immunoblots of the acrosomal fraction separated by reducing SDS-PAGE revealed a single anti-HEP64 immunoreactive band of 64 kDa (Fig. 2, lane 7), and no immunoreactive bands were noted with identical dilutions of preimmune serum (Fig. 2, lane 8).
The carbohydrate content of HEP64 was assessed using acid deglycosylation followed by SDS-PAGE and immunoblot analysis. Compared to native HEP64 (Fig. 3, lane 1) the deglycosylated form migrated with an apparent molecular weight of 52 kDa (Fig. 3, lane 2). This suggests that HEP64 possesses approximately 12 kDa of oligosaccharide moieties. To determine if HEP64 was comprised of charge variant isoforms, sperm fractions were separated by two-dimensional PAGE and subjected to immunoblot analysis. Blots stained with anti-HEP64 exhibited a charge train of 12 to 13 distinct spots with isoelectric points ranging between pH 8.5 to 6.5 (Fig. 4A). No stained bands were seen with preimmune serum (Fig. 4B).
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Immunolocalization of HEP64
To determine if HEP64 exhibited a discrete localization pattern, cauda epididymal spermatozoa were subjected to a variety of fixation and permeabilization regimens and then immunostained with anti-HEP64. Surprisingly, most cauda epididymal spermatozoa exhibited no fluorescence staining, and companion phase-contrast images demonstrated that the unstained spermatozoa appeared morphologically normal (Fig. 5, A, A', C, C', and D, D'). However, a subset of the cauda epididymal spermatozoa population did stain with anti-HEP64 serum. Counts of at least 200 spermatozoa from the cauda epididymides of three animals revealed staining on 9–13% of the total sperm population. Positive staining was noted on sperm aggregates that were intensely fluorescent and appeared embedded within immunostained amorphous material (Fig. 5, A and A'). Many of the aggregated spermatozoa stained along their entire length, and by phase-contrast microscopy these spermatozoa usually appeared morphologically abnormal. Occasional single spermatozoa also exhibited positive staining with anti-HEP64; these spermatozoa exhibited variable staining patterns and some were stained along their entire length, but frequently only discrete segments were stained (Fig. 5, A' and D'). For example in spermatozoa with positively stained anterior heads, the acrosome was usually misshapen or absent; in the most extreme examples spermatozoa or sperm fragments were embedded in a shroud of material that exhibited intense staining with immune serum (Fig. 5, C and C'). No detectable differences in the staining pattern were noted if sperm were permeabilized with Triton X-100 prior to formaldehyde fixation (data not shown). No stained spermatozoa or sperm aggregates were detected in control samples immunostained using preimmune serum (Fig. 5, B and B'). Extensive examination of caput sperm samples stained with anti-HEP64 failed to reveal any sperm with detectable staining (not shown). These data demonstrate that HEP64 selectively associates with damaged spermatozoa and suggest that it exhibits a region-specific expression pattern in the epididymis.
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The region-specific expression pattern of HEP64 was defined by immunostaining epididymal cryosections. In the distal cauda epididymidis the tubule lumen exhibited discrete foci of intense staining; these were comprised of single or aggregated spermatozoa. No staining was detected within the principal cells of the distal cauda epididymidis or in the surrounding stroma (Fig. 6, A and A'). In the proximal cauda region, staining of focal aggregates within the tubule lumen was also apparent; however, most principal cells also exhibited positive staining in the Golgi region (Fig. 6, B and B'). The overall luminal staining in the proximal cauda region was less intense than the distal cauda (compare Fig. 6, A' and B'). In the corpus region occasional clusters of principal cells with positively stained Golgi were noted, while neighboring principal cells exhibited no detectable Golgi staining; in comparison to both the proximal and distal cauda regions, the lumen of the corpus epididymidis also exhibited diminished staining, although some stained spermatozoa and/or sperm aggregates were noted (Fig. 6, C and C'). In the caput region no staining of either the epithelium or tubule lumen was detectable (Fig. 6, D and D'). These data suggest that HEP64 is produced by principal cells of the corpus and proximal cauda regions of the epididymis and that it is secreted to the tubule lumen where it becomes associated with a restricted sperm population.
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Immunoelectron microscopy was utilized to examine the association of HEP64 with cauda epididymal spermatozoa. Intact spermatozoa exhibited no specific labeling with anti-HEP64. In contrast, degenerating spermatozoa were coated with a layer of amorphous material which labeled with anti-HEP64 (Fig. 7). No labeling of any spermatozoa was detected with equivalent dilutions of preimmune serum (not shown).
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Immunoblot Analysis of HEP64 in Spermatozoa and Epididymal Fluid
Because immunocytochemistry suggested that HEP64 is a secretory protein of principal cells, immunoblot analyses were employed to determine if sperm-associated HEP64 is tightly bound. Total lysates of cauda spermatozoa revealed a single 64-kDa immunoreactive band (Fig. 8, lane 1). Samples of cauda epididymal spermatozoa were extracted with Triton X-100 under either isotonic or high salt (0.5 M NaCl) extraction conditions and then centrifuged. Immunoblot analysis of the supernatant and pellet fractions revealed that most HEP64 remained associated with the pellet fraction under both extraction conditions, and only a minor amount was noted in the supernatant fraction following high salt extraction (Fig. 8, lanes 2–5). This demonstrates that HEP64 is firmly bound to spermatozoa and resists extraction by nonionic detergent and elevated ionic strength.
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Finally, to assess whether HEP64 is present in the soluble as well as the insoluble fraction of the epididymal luminal contents, immunoblot analysis was performed comparing crude epididymal luminal fluid and epididymal spermatozoa from the caput and cauda regions of the epididymis. No immunoreactive bands were noted in epididymal fluid or spermatozoa of the caput epididymidis (Fig. 9A, lanes 1 and 2). However both luminal fluid and spermatozoa of the cauda epididymidis exhibited a single immunostained band of 64 kDa (Fig. 9A, lanes 3 and 4). This confirmed the regional expression of HEP64 demonstrated by immunocytochemistry and also indicated that, in addition to sperm-associated HEP64, soluble HEP64 is present in the luminal fluid. To determine if luminal fluid as well as whole sperm-associated HEP64 are assembled into high molecular weight, disulfide-linked complexes, immunoblot analyses were also performed on nonreduced samples separated by high porosity SDS-PAGE. The HEP64 of both the cauda epididymal fluid and cauda spermatozoa migrated as a doublet of 280 and 260 kDa; both samples also exhibited a minor band of 200 kDa that may represent an oligomeric intermediate (Fig. 9B, lanes 1 and 2); no specific bands were noted in parallel lanes immunostained with preimmune serum (Fig. 9B, lanes 3 and 4). These data suggest that HEP64 is secreted from epididymal principal cells as a disulfide cross-linked oligomeric complex and demonstrate that monomeric HEP64 is not detected in epididymal fluid.
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DISCUSSION
Even though the epididymis provides an environment responsible for the post-testicular maturation and survival of spermatozoa [1], a population of degenerating sperm is typically noted within the tubule lumen [19–21]. During characterization of a sperm subfraction, we unexpectedly identified a glycoprotein, termed HEP64, secreted by the principal cells of the corpus and cauda epididymidis that selectively binds the damaged/dead sperm population. It is possible that this coating material plays a protective function to shield the viable sperm population and/or epididymal epithelium from the degenerating spermatozoa.
The biochemical data demonstrate that within the epididymal lumen both soluble and sperm-associated HEP64 are incorporated into a disulfide cross-linked polymeric complex. Nonreducing SDS-PAGE reveals two HEP64 bands of 260 and 280 kDa, and no 64-kDa band is detected. When the high molecular weight complexes are subsequently fractionated by reducing SDS-PAGE, only a single band of 64 kDa is noted, suggesting that the high molecular weight complexes represent homopolymeric aggregates of at least four to five 64-kDa subunits. Two-dimensional PAGE reveals a complex charge-variant pattern of HEP64 monomers with 12–13 isoforms that may represent glycosylation variants. Whether other post-translational modifications such as sulfation, which generates the charge variants of SGP-2 present in the epididymis [14, 34], or phosphorylation also contribute to the charge heterogeneity of HEP64 remains to be investigated. Functionally, it will be crucial to determine if the different HEP64 isoforms exhibit similar or different ligand-binding specificity, as it is presently not clear how HEP64 selectively associates with different sperm organelles.
The HEP64 glycoprotein is a constituent of the thick shroudlike coating of amorphous material surrounding aggregates of degenerating spermatozoa identified in earlier electron microscopic studies [21]. Presently, it is uncertain whether the coating material is comprised only of HEP64; if so this would suggest that the HEP64 oligomers self-associate into higher order complexes. Alternatively, it is possible that other polypeptide(s) interact with HEP64 to form the coating shroud of dead spermatozoa. Future studies will address these possibilities and could identify other epididymal proteins targeted for specific interaction with dead spermatozoa.
Previous studies of human spermatozoa have suggested that dimeric seminal plasma clusterin, termed SP-40,40 in humans, is associated with abnormal spermatozoa [35, 36]; however, another study using the same monoclonal antibodies demonstrated a different localization pattern on human spermatozoa and concluded that SP-40,40 is associated with viable spermatozoa [37]. The biochemical properties, temporal expression pattern, and specific association of HEP64 is distinct from that of SGP-2, which is a disulfide-linked dimer of two 40-kDa subunits and migrates at 70 kDa by nonreducing SDS-PAGE [15, 38]. Moreover SGP-2 is a major secretory product of Sertoli cells and the proximal epididymis in rats [15, 38, 39], and it associates with the normal sperm population within the epididymis. Also in the distal regions of the epididymis luminal SGP-2 levels are considerably lower due to removal by epithelial endocytotic activity [15, 38], whereas the highest levels of HEP64 protein are detected in the distal cauda epididymidis, and none is detected in the caput region. Nonetheless whether some SGP-2 does associate with the HEP64-containing coating on nonviable cauda epididymal hamster spermatozoa will be addressed in future studies.
Several critical questions regarding the mechanism of action of HEP64 remain to be resolved. Presently, we do not know if HEP64 only binds dead sperm or whether it could also function to promote the death of damaged spermatozoa. Another unresolved question is to define the mechanism by which HEP64 specifically binds damaged sperm. Electron microscopic immunocytochemistry reveals that HEP64 does not bind to the sperm plasma membrane but instead associates with segments of the spermatozoon lacking an intact plasma membrane. It binds to a variety of exposed sperm organelles including the acrosome, the postacrosomal segment, the midpiece mitochondrial sheath, and the principal piece fibrous sheath. Ultimately, single spermatozoa or sperm aggregates are completely coated by an amorphous shroud containing HEP64. Although HEP64 may directly bind exposed sperm organelles, another possibility is that its interaction is indirect via other complexed polypeptide(s). Future identification of proteins complexed to HEP64 as well as the testing of the ability of purified HEP64 to bind various sperm proteins and organelles of demembranated spermatozoa should provide direct evidence for the molecular basis of its recognition of damaged spermatozoa.
Like many epididymal principal cell secretory proteins [1, 8], HEP64 is expressed in a restricted region of the epididymis, and to date the mechanisms regulating region-specific epididymal protein expression remain unresolved. The expression pattern of HEP64 correlates with the development of sperm-fertilizing capacity, but highest luminal expression levels appear in the distal cauda region of the epididymis, which is specialized for sperm storage. Thus, its expression may be critical for maintenance of a viable sperm population. Factors regulating HEP as well as its molecular analysis will be addressed in future studies. Particularly intriguing will be to determine if manipulations that disrupt sperm maturation or epididymal storage capacity and result in reduced sperm viability also promote increased expression of this sperm death-associated protein.
FOOTNOTES
First decision: 15 March 2000.
1 Supported by NIH grant HD20419. 
2 Correspondence. FAX: 615 343 4539; subir.nag-das{at}mcmail.vanderbilt.edu
Accepted: June 15, 2000.
Received: February 15, 2000.
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