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Biol Reprod 2004, 10.1095/biolreprod.104.029975
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BIOLOGY OF REPRODUCTION 71, 1128–1134 (2004)
DOI: 10.1095/biolreprod.104.029975
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Zonadhesin Assembly into the Hamster Sperm Acrosomal Matrix Occurs by Distinct Targeting Strategies During Spermiogenesis and Maturation in the Epididymis1

Gary E. Olson2,3, Virginia P. Winfrey3, Ming Bi4, Daniel M. Hardy4, and Subir K. NagDas3

Department of Cell and Developmental Biology,3 Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Department of Cell Biology and Biochemistry,4 Texas Tech University Health Sciences Center, Lubbock, Texas 79430


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.

acrosomal matrix, acrosome, acrosome biogenesis, acrosome reaction, sperm, spermatid, sperm maturation, testis, zonadhesin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian fertilization, the capacitated spermatozoon establishes contact with the cumulus/oocyte complex within the ampulla of the oviduct. There, it penetrates between the cells of the cumulus oophorus, then through the zona pellucida (ZP), to reach the oocyte plasma membrane [1, 2]. The species-specific adhesion of the spermatozoon to the ZP and the initiation of the sperm acrosome reaction are key steps in the fertilization process. The species specificity of sperm adhesion is reflected in a striking interspecies variation in the structures of candidate sperm-recognition molecules [3]. At present, despite much investigation, the temporal sequence of specific molecular interactions between gametes remains poorly understood [4, 5]. In part, this reflects the uncertainty of the functions and the subcellular locations of the several candidate sperm molecules proposed to function in ZP adhesion.

The prevailing view of sperm-egg interaction is that receptors or adhesion molecules in the periacrosomal plasma membrane of the intact spermatozoon mediate adhesion to the ZP and that this adhesion then initiates the acrosome reaction that is necessary for ZP penetration [4, 6]. However, this concept is based largely on results of in vitro light-microscopic analyses of sperm interaction with cumulus-free eggs. These studies clearly demonstrate that sperm-ZP adhesion promotes the acrosome reaction. On the other hand, several in vivo studies indicate that acrosomal modifications, and even the membrane fusion events of the acrosome reaction, are initiated in at least some of the spermatozoa penetrating the cumulus and thus before contact with the zona pellucida [2, 7–11]. In these spermatozoa, a binding element located within the acrosome could contribute to gamete interactions at the time of the primary ZP adhesion event. Furthermore, even when the acrosome reaction is initiated after intact spermatozoa make contact with the zona pellucida, binding elements within the acrosome may function to establish stable adhesion.

Zonadhesin is a large multidomain protein that was originally identified in a porcine sperm membrane preparation based on its high-affinity and species-specific binding to the egg ZP [12]. Subsequent analyses demonstrated a testis-specific expression of zonadhesin mRNA and the protein was shown to reside specifically within the acrosomal segment of the mature spermatozoon [13, 14]. Zonadhesin cDNAs have been cloned from pig [13], mouse [14], human (unpublished results) and rabbit [15] testes, and the deduced proteins each possess a predicted transmembrane segment and a short cytoplasmic tail, with the bulk of the protein residing in a large predicted extracellular region. Zonadhesin's ZP-binding activity, restricted localization, and membrane association suggested that it functions as a sperm surface receptor for the ZP [13, 14]. However, we have recently demonstrated that zonadhesin in porcine spermatozoa is located within the acrosome and not on the plasma membrane [16]. Here, we show further that, in hamster spermatozoa, zonadhesin is a component of a stable element of the acrosomal content termed the acrosomal matrix. Incorporation of zonadhesin into the acrosomal matrix is shown to involve distinct targeting events during spermiogenesis and posttesticular sperm maturation in the epididymis. These observations raise important questions as to the nature of the primary ZP adhesion event in mammalian fertilization and suggest that the acrosomal matrix has a direct role in the high-avidity adhesion of spermatozoa to the ZP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Care and use of animals conformed to National Institutes of Health guidelines for humane animal care and use in research. Animal protocols were approved by the Institutional Animal Care and Use Committee. Adult male golden hamsters were housed in an AAALAC-approved facility on a 14L:10D cycle and with free access to food and water. Animals were killed by CO2 asphyxiation and their testes and epididymides were removed immediately and used in the following procedures.

Acrosomal Matrix Isolation

An enriched acrosomal matrix fraction was isolated from cauda epididymal spermatozoa using an established protocol [17, 18]. Sperm pellets were resuspended with at least 10 volumes of ice-cold extraction buffer, composed of 0.1% Triton X-100 in TNI (TNI = 150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 2 mM benzamidine, and 1 µg/ml each of leupeptin and pepstatin), and then immediately centrifuged at 1500 x g for 10 min. The sperm pellet was resuspended in fresh extraction buffer and incubated for 30 min at 4°C and then diluted with four volumes of a solution of 45% Percoll, 0.25 M sucrose, 0.1% Triton X-100, 25 mM Tris-HCl, pH 7.5, and centrifuged at 60 000 x g for 35 min in a Beckman 60Ti rotor. The acrosomal matrix fraction banded in the upper part of the gradient and it was collected, diluted with extraction buffer, and pelleted by centrifugation at 100 000 x g for 60 min in a Beckman SW41 rotor.

To test the solubility properties of zonadhesin, the acrosomal matrix fraction was extracted for 30 min at room temperature in 1% SDS in TNI and then centrifuged at 100 000 x g at 25°C for 30 min. The supernatant and pellet fractions were collected for immunoblot analyses.

SDS-PAGE and Immunoblotting

Acrosomal matrix fractions were solubilized in SDS sample buffer for 5 min at 95°C in the presence or absence of 5% dithiothreitol for reducing or nonreducing PAGE, respectively. SDS-PAGE was performed on 5–15% or 3–19% acrylamide gradient gels [19] using the protein loads specified in the figure legends. Gels were either stained with Coomassie blue or polypeptides were electrophoretically transferred to polyvinylidene fluoride membranes for immunoblot analysis [20]. Membranes were blocked with PBST (0.1% Tween 20, 150 mM NaCl, 20 mM sodium phosphate, pH 7.6) containing 5% goat serum, 5% nonfat dry milk, and 2.5% BSA. Blots were then incubated in antizonadhesin or control serum diluted 1: 2500 in PBST with 1% goat serum (PBS-GS) for 1 h. This primary antibody, prepared in rabbits against porcine sperm zonadhesin holoprotein [21], reacts with the spermatozoa of several mammals tested. The blots were then washed in PBST-GS and then incubated in an affinity-purified horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA) diluted 1:5000 in PBST-GS. Following several washes in PBST, immunoreactive bands were visualized by color development with diaminobenzidine and H2O2.

Coomassie blue-stained bands were excised from the gels and subjected to trypsin digestion and sequence analysis by the Harvard Microchemistry Facility using microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry on a Finnigan LCQ DECA quadropole ion trap mass spectrometer (µLC-MS-MS). Peptide sequences were identified using the National Center for Biotechnology Information BLAST programs [22].

Immunocytochemistry

Hamster spermatozoa were fixed in 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, and allowed to attach to poly-L-lysine coated coverglasses. Adherent spermatozoa were either rinsed in 0.1 M ammonium acetate or permeabilized at –20°C in absolute acetone and then air dried. Spermatozoa were then incubated in 0.1% Triton X-100 in TN (150 mM NaCl, 0.1% Tween 20, and 25 mM Tris-HCl, pH 7.5), blocked for 1 h in TN containing 2.5% BSA and 5% donkey serum and incubated for 1 h in anti-porcine zonadhesin or nonimmune rabbit serum diluted 1:2000 in blocking solution. After several rinses in TN, specimens were incubated in a secondary antibody of Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) diluted 1:1000 in blocking solution. The slides were then rinsed at least three times in TN and both experimental and control specimens were examined with a Zeiss Axiophot and photographed using identical exposure times.

Immunoelectron Microscopy

For postembedding immunolabeling, testes and epididymal spermatozoa were fixed on ice with 4% formaldehyde, 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Samples were then rinsed in buffer, dehydrated through an ethanol series, and embedded in LR White Resin (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were mounted on nickel grids and stained as for immunofluorescence except that gold-conjugated affinity-purified secondary antibodies were used (Amersham Inc., Arlington Heights, IL). Primary antisera were diluted 1: 100, secondary antibody was diluted 1:50, and incubation times of 1 h at room temperature were used for each. After immunolabeling, the sections were washed in PBS, fixed with 1% glutaraldehyde, rinsed with water, and stained with uranyl acetate and lead citrate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Zonadhesin as a Constituent of the Acrosomal Matrix

The purified acrosomal matrix fraction isolated from hamster cauda epididymal spermatozoa was resolved into a limited number of polypeptides by SDS-PAGE (protein disulfides reduced; Fig. 1). The major set of polypeptides with Mr between 22 000 and 29 000 were previously identified as processing variants of a common precursor polypeptide [18], but the identities of the other major acrosomal matrix components have not been reported. Peptide mapping and sequencing of the Mr 215 000 polypeptide by lethal concentration MS-MS identified three constituent peptides (Fig. 1) identical to sequences in mouse zonadhesin [14], a sperm protein with zona pellucida binding activity [12]. Peptide 1 mapped to the MAM domain of mouse zonadhesin, whereas peptides 2 and 3 mapped to its repetitive D domains. We previously showed that antisera to the porcine zonadhesin holoprotein bound to disulfide nonreduced zonadhesin [16, 21]. On immunoblots of the hamster acrosomal matrix fraction, the holoprotein antisera recognized a major acrosomal matrix polypeptide that migrated at Mr 300 000 (protein disulfides not reduced; Fig. 2, lanes 1 and 4), confirming the presence of zonadhesin. Most zonadhesin was not released from the acrosomal matrix fraction by extraction in 1% SDS at room temperature, suggesting that it is a structural component of the matrix (Fig. 2, lanes 2, 3, 5, 6).



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  		FIG. 1. Reducing SDS-PAGE stained with Coomassie blue reveals limited polypeptide composition of the purified acrosomal matrix fraction (60 µg protein) of hamster cauda epididymal spermatozoa. AM29 and 22, representing the major matrix polypeptides, are processed from a common precursor polypeptide [18]. LC-MS-MS identification of tryptic digests of the Mr 215 000 polypeptide yielded the three peptides shown that matched mouse zonadhesin



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  		FIG. 2. Coomassie blue stained gel (lanes 1–3) and antizonadhesin-stained immunoblots (lanes 4–6) of the purified acrosomal matrix fraction separated by nonreducing SDS-PAGE. The total acrosomal fraction (lanes 1 and 3) contains a prominent Coomassie blue-stained band of Mr ~300 000 (lane 3, 75 µg protein load) that is recognized by antizonadhesin (lane 4, 35 µg protein load). Extraction of the acrosomal matrix fraction in 1% SDS and centrifugation at 100 000 x g to obtain supernatant (SDS-S, lanes 2 and 5) and pellet (SDS-P, lanes 3 and 6) fractions show that most zonadhesin resists solubilization and remains in the pellet fraction with AM29 and AM22. Lanes 2 and 3 represent supernatant and pellets derived from 75 µg of acrosomal matrix protein and lanes 5 and 6 from 35 µg of acrosomal matrix protein

Light and electron microscopic immunocytochemistry confirmed the association of zonadhesin with the acrosomal matrix and defined its distribution. Immunofluorescence staining of spermatozoa that were formaldehye-fixed and then permeabilized with Triton X-100 or acetone localized zonadhesin to the apical and principal segments but not the equatorial segment of the acrosome; no detectable staining was associated with other sperm structures (Fig. 3, A and B) and no fluorescence was observed in control specimens stained with nonimmune rabbit serum (not shown). Immunofluorescence staining of spermatozoa that were first Triton extracted and then fixed with formaldehyde localized zonadhesin specifically to the crescent-shaped, detergent-insoluble acrosomal cap (Fig. 3, C and D). In frontal views, the acrosomal caps exhibited a slit-like opening along their dorsal midlines; no zonadhesin was detected on the sperm head after detachment of the acrosomal cap (Fig. 3, C and D). To define the distribution of zonadhesin in the acrosomal matrix, antizonadhesin was employed for immunoelectron microscopic analysis of Triton X-100-extracted cauda spermatozoa. Triton extraction removes soluble acrosomal constituents, but the stable acrosomal matrix infrastructure remains intact and comprises two structurally distinct contiguous domains, termed m1 and m2 [17, 23]. Both the m1 and m2 domains of the acrosomal matrix labeled intensely with antizonadhesin (Fig. 3, E and F). After release of the acrosomal matrix, no zonadhesin was detected over the anterior sperm head nor was labeling detected in the Triton-insoluble components of the equatorial segment or on other sperm organelles.



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  		FIG. 3. A–D) Matched phase contrast (A, C) and fluorescence (B, D) images of cauda epididymal spermatozoa immunostained with antizonadhesin. A and B) Spermatozoa that were formaldehyde-fixed and then permeabilized using Triton X-100. Note the specific staining of the acrosomal cap (ac) representing the apical and principal segments and the absence of staining over the remainder of the spermatozoon. C and D) Immunofluorescence of spermatozoa extracted with Triton X-100 before fixation. Note that the detergent-resistant acrosomal cap (ac) displays a midline opening in frontal views. E, F) Postembedding immunoelectron microscopy of Triton X-100-extracted cauda spermatozoa shows that zonadhesin localizes specifically to the m1 and m2 domains of the acrosomal matrix. No zonadhesin is detected over the nucleus (n). mp, Midpiece; pp, principal piece

Zonadhesin Targeting to Specific Acrosomal Regions During Spermiogenesis

In both Golgi phase and cap-phase round spermatids, zonadhesin localized to the acrosomal membrane and appeared evenly distributed between the nascent inner and outer membrane domains (Fig. 4A). No specific labeling was detected in the acrosomal granule or on the spermatid plasma membrane. In cap-phase spermatids, a comparable labeling intensity was detected in the acrosomal membrane surrounding the acrosomal granule and in the thin membrane fold of the head cap (Fig. 4A). In late spermatids, zonadhesin was detected throughout the length of the acrosome, but it exhibited a different distribution than in round spermatids (Fig. 4, B–D). In the apical and principal segments, zonadhesin was associated exclusively with the outer acrosomal membrane, which is distinguished by an electron-dense assembly adherent to its luminal surface (Fig. 4C). Both zonadhesin and the electron-dense membrane coating were absent, however, from the dorsal sagittal midline of the outer acrosomal membrane (Fig. 4, B and C). No zonadhesin was detected either on the inner acrosomal membrane or within the anterior acrosome (Fig. 4, B and C). In contrast, in the developing equatorial segment, zonadhesin labeling was less intense and most gold particles were associated with the acrosomal contents. No zonadhesin was detected at the posterior margin of the equatorial segment, where the inner and outer acrosomal membranes had become closely paired in a parallel configuration (Fig. 4D). Control specimens stained with nonimmune serum displayed no specific labeling.



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  		FIG. 4. Postembedding immunoelectron microscopy showing the acrosomal localization of zonadhesin in spermatids. A) In round spermatids, zonadhesin localizes to the perimeter of the acrosome and is associated with the developing outer (oa) and inner (ia) acrosomal membranes. Note that zonadhesin is restricted from the acrosomal granule (ag) but it is present in the acrosomal membranes that extend laterally to form the head cap (arrows). B–D) Electron micrographs showing zonadhesin distribution in late spermatids. B) Low magnification micrograph showing that zonadhesin is present along the full length of the acrosome; however, note that, anteriorly, zonadhesin is restricted to the perimeter of the acrosome, whereas it is detected throughout the developing equatorial segment (eq, between arrowheads). Note also the absence of zonadhesin at the dorsal margin of the acrosome (*). C) High magnification view of anterior acrosome of late spermatid showing that zonadhesin localizes to the outer acrosomal membrane (oa), which is distinguished by an adherent electron-dense lamina and that no labeling is now detected on the inner acrosomal membrane (ia). Note the absence of both zonadhesin and the electron-dense lamina at the sagittal midline of the acrosome (*). S, Sertoli cell cytoplasm. D) In comparison with the anterior acrosome, the equatorial segment (eq, between arrowheads) of late spermatids shows reduced labeling for zonadhesin, which is distributed throughout its interior. Note the absence of zonadhesin at the posterior margin of the equatorial segment where the inner and outer acrosomal membranes are in close apposition. n, Nucleus; pa, postacrosomal segment

Zonadhesin Redistribution During Epididymal Maturation

In caput spermatozoa, most zonadhesin localized toward the periphery of the apical and principal segments and appeared to be associated with an electron-dense lamina beneath the outer acrosomal membrane, although some labeling was also detected in the acrosomal contents immediately adjacent to the lamina (Fig. 5, A and B). This lamina appeared more prominent in the ventral aspect of the acrosome, where the m2 domain of the acrosomal matrix was forming. Compared with caput spermatozoa, those from the cauda displayed a further redistribution of zonadhesin, which was mostly now separated from the outer acrosomal membrane and superimposed on both the m1 and m2 regions of the acrosomal matrix (Fig. 5C). In a few spermatozoa that exhibited slight acrosomal swelling, the matrix, which remained associated with the outer acrosomal membrane, labeled intensely for zonadhesin, while the more interior contents displayed little labeling (Fig. 5D). In no case was zonadhesin detected either along a dorsal midline seam that extended for most of the length of the apical and principal segments (Fig. 5, A–D) or in the equatorial segment (Fig. 5E).



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  		FIG. 5. Immunoelectron microscopy showing zonadhesin localization in spermatozoa from the proximal caput (A), distal caput (B, E) and cauda (C, D) regions. In cross-sections A–D, the asterisk (*) represents the dorsal midline of the acrosome that possesses no detectable zonadhesin. A, B) In caput spermatozoa, most zonadhesin is detected at the acrosome periphery, colocalizing with the developing dorsal m1 and ventral m2 acrosomal matrix; note that zonadhesin is not as closely localized to the outer acrosomal membrane as in late spermatids (compare with 4C). C) In cauda spermatozoa, zonadhesin localizes throughout the m1 and m2 domains of the acrosomal matrix. D) An abnormally swollen acrosome of a cauda spermatozoon showing that zonadhesin is localized to the m1 and m2 acrosomal matrix but is absent in the more internal acrosomal compartment. E) Longitudinal section of head of distal caput spermatozoon showing the absence of detectable zonadhesin in the equatorial segment (eq) and its localization to the acrosomal matrix (m2) in the anterior acrosome. The arrowhead shows the junction of the equatorial (eq) and the postacrosomal (pa) segments. n, Nucleus; mp, midpiece; pp, principal piece; ia, inner acrosomal membrane; pn, perinuclear material


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite intense study, the repertoire of molecular interactions responsible for the species-specific adhesion between the spermatozoon's acrosomal segment and the ZP remain poorly understood. After ZP adhesion and completion of the acrosome reaction, the spermatozoon penetrates the ZP, while the detached remnant of the acrosomal cap remains affixed to the ZP surface [2, 24–26]. In the hamster, the major structural elements of the detached acrosomal cap include the hybrid membrane vesicles, generated by fusion of the outer acrosomal membrane and plasma membrane, and the stable acrosomal matrix [24, 25, 27], but the array and location of ZP-binding proteins in this complex are not established. In this study, we demonstrate that zonadhesin, the only sperm protein known to bind the ZP in a species-specific manner [12, 13], is a constituent of the acrosomal matrix. This finding suggests that, like invertebrates, where intra-acrosomal components play a key role in egg adhesion [28], the acrosomal matrix of mammalian spermatozoa may also play a direct role in sperm adhesion to the ZP.

Acrosome biogenesis occurs throughout spermiogenesis and is completed during sperm maturation in the epididymis [23, 29]. Our results reveal that the targeting and incorporation of zonadhesin into the acrosomal matrix involves distinct steps in both of these developmental processes. Whereas zonadhesin exhibits a uniform distribution in the acrosomal membrane of round spermatids, it exhibits two fates during spermatid elongation. First, in the apical and principal segments, it disappears from the inner and becomes segregated to the outer acrosomal membrane. Loss of zonadhesin from the inner acrosomal membrane probably results from its diffusion within the plane of the membrane, as none is detectable then within the contents of the anterior acrosome; moreover, its segregated distribution may then be maintained by an association with the scaffold-like structure underlying the outer acrosomal membrane. Second, in late spermatids, the first indication of a nonmembrane-anchored zonadhesin becomes evident within the contents of the nascent equatorial segment, and this pool of zonadhesin is subsequently eliminated from the equatorial segment as the inner and outer acrosomal membranes become zipped together in a parallel configuration. The final phase occurs in epididymal spermatozoa, where zonadhesin is released from the outer acrosomal membrane and begins to redistribute, such that it becomes restricted to the m1 and m2 regions of the acrosomal matrix. In the pig and rabbit, zonadhesin is known to undergo extensive posttranslational proteolytic processing and disulfide bond formation during spermiogenesis and sperm maturation in the epididymis resulting in lower molecular weight, disulfide-linked oligomers [15, 21]. Because zonadhesin is present within the acrosome, not the sperm surface, its extracellular segment faces the acrosomal lumen, and so proteolytic processing must release this membrane-anchored segment, permitting its redistribution to the acrosomal contents. Our ultrastructural data clearly show that zonadhesin undergoes a dynamic redistribution from a membrane-associated to a matrix-associated location. Whether this requires posttranslational processing events other than proteolysis-dependent loss of membrane anchoring remains to be determined.

In late spermatids and caput epididymal spermatozoa, both zonadhesin and the submembranous scaffolding of the outer acrosomal membrane are conspicuously absent from a sagittal midline seam of the maturing acrosome. This seam creates the opening that is apparent in the Triton-insoluble acrosomal cap of cauda spermatozoa, suggesting that zonadhesin confers a structural integrity to the acrosomal matrix. We propose that this seam also becomes the opening that is apparent in the acrosomal cap of zona-adherent acrosome-reacting spermatozoa and through which the spermatozoon passes to enter the zona [25]. Thus, even following its release from the sperm head, the acrosomal cap still functions as a collar-like tether for the penetrating spermatozoon [2]. It is also possible that, as the spermatozoon passes through the acrosomal cap, its surface is enzymatically modified by hydrolases bound to the acrosomal matrix [30, 31] and that such modification contributes to the development of its capacity to fuse with the egg plasma membrane [1].

Although zonadhesin is the only protein demonstrated to exhibit species-specific binding activity [12], it is not the only acrosomal protein suggested to function in ZP adhesion. Others include proacrosin/acrosin [32, 33], mouse and bovine SP10 [34], which are orthologs of hamster acrosomal matrix AM22 and AM29 [18, 23], guinea pig MC41 [35], and mouse sp56 [36]. Although it was first proposed that mouse sp56 acts as a sperm surface receptor for the ZP [37, 38], both it [39] and its guinea pig ortholog, AM65 [36], prove to reside exclusively within the acrosomal contents. These data support a role for the stable acrosomal matrix and its associated proteins in adhesion to the egg investments. Whether the acrosomal components function in primary or secondary binding events clearly depends on where the acrosome reaction is initiated. In eutherian mammals, both the progesterone, present in follicular fluid and probably the cumulus, and the ZP promote the sperm acrosome reaction [4, 40, 41]. Because only acrosome loss and not the membrane fusion events that initiate the acrosome reaction are typically detected with the light microscope, the normal timing of the induction step is uncertain. However, several in vivo studies have shown that the acrosome may respond as potential fertilizing spermatozoa pass through the cumulus cells [2, 42, 43], perhaps induced to do so by the high levels of progesterone present. This raises uncertainty as to whether the membrane fusion events of the acrosome reaction are initiated before or only upon sperm adhesion to the ZP. An attractive model, termed the transition state hypothesis, proposes that focal fusions of the plasma and outer acrosomal membrane of capacitated spermatozoa expose intra-acrosomal components that function in ZP adhesion [29]. At the least, these data suggest that the current concept of sperm-ZP interactions, in which fertilizing spermatozoa are seen as adhering to the ZP in an acrosome-intact state, needs revisiting. Clearly, high-resolution analyses of in vivo and in vitro models are required to assess the status of the acrosome during sperm penetration through the cumulus and at the time of ZP adhesion.


   FOOTNOTES
 
1 Supported by HD-35166 (D.M.H.) and HD-20419 (G.E.O.). Back

2 Correspondence: Gary E. Olson, Department of Cell and Developmental Biology, 1161 21st Ave. S, Room T-2208 MCN, Vanderbilt University, Nashville, TN 37232. FAX: 615 343 4539; gary.olson{at}vanderbilt.edu Back

Received: 23 March 2004.

First decision: 15 April 2004.

Accepted: 20 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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