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Regular Article |
a Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Miyazaki 8891692, Japan
ABSTRACT
Equatorin is a sperm head equatorial protein, possibly involved in sperm-oocyte fusion (Toshimori et al., Biol Reprod 1998; 59:22–29). In the present work, we have shown that equatorin contained in the posterior acrosome is detectable only after spontaneous or induced acrosome reactions following fixation and permeabilization, but not in intact spermatozoa. The presence of protease inhibitors during sonication or ionophore treatments does not inhibit the exposure of the antigenic epitope. The zona-penetrated spermatozoa lying in the perivitelline space display equatorin, similar to those of the acrosome-reacted ones. After sperm-egg fusion during in vitro fertilization (IVF), the equatorin dissociates from the sperm head equatorial region and remains at the vicinity of the decondensing male pronuclei. During pronuclear apposition stage, it is pushed away from the pronuclei, possibly by the perinuclear microtubules. After first cleavage, equatorin is inherited by one of the proembryonic cells. The residual equatorin disappears after the second cleavage. Microinjected whole spermatozoa or sperm heads into the MII stage oocytes display equatorin similar to those of the perivitelline sperm. After activation, it dissociates from the sperm nuclei in a similar manner as during IVF. The mode of equatorin degeneration during fertilization is similar to those of the sperm tail components or mitochondria, but different from those of the membrane associated proteins.
acrosome reaction, embryo, fertilization, in vitro fertilization, sperm
INTRODUCTION
Besides nuclei, the functions of various ancillary organelles of spermatozoa are crucially important to ensure successful fertilization. The tail propels the spermatozoan forward; the acrosomal enzymes clear the way through cumulus cells and zona pellucida [1]; the mitochondria of the midpiece supply energy; the neck region harbors centrosomes, the function of which are essential for pronuclear movement/apposition [2]; and the sperm head equatorial region provides a molecular machinery to introduce the spermatozoan into the oocyte by fusing with the oolemma [3]. The functions of most of the extranuclear organelles are concluded after fusion, when the entire spermatozoan except the anterior acrosomal sac is incorporated into the oocyte; these organelles seem to degenerate in the oocyte. Disintegration of the sperm tail, mitochondria, and perinuclear theca have been shown in various studies with electron and immunofluorescent light microscopy [4–12].
The equatorial region of the sperm head is marked with distinctive features that are different from the other domains of the spermatozoan. It consists of a narrow membranous pouch (about 45 nm wide) formed by a posterior extension of the acrosomal sac, which is why it is also known as the posterior acrosome (Fig. 1). Most hydrolytic enzymes and other acrosome marker molecules seem to be restricted to the anterior, saclike region of the acrosome [13–19]. The composition of the posterior acrosome, on the other hand, is less characterized. Among the few constitutive molecules found in this region, equatorin, as recognized by the monoclonal antibody (mAb) MN9, has been systematically studied [20, 21]. In mouse spermatozoa it is one or more proteins 38–48 kDa in length localized in the inner matrix of the posterior acrosome. It has also been detected as equatorial bands in rat, hamster, human [20], guinea pig, and bull spermatozoa (unpublished observations). Other proteins are also present in the sperm equatorial region, as shown by immunofluorescent and electron microscopy. Among them, the proteins recognized by antibodies M29 [22], M1 [23], G11, and G13 [24] appear after the acrosome reaction, whereas oscillin is revealed in permeabilized spermatozoa [25]. A set of fusion-related soluble N-ethylmalameide-sensitive factor attachment protein receptors (SNAREs) also occur in the equatorial region of a wide variety of mammalian spermatozoa [26]. Ultrastructurally, the outer and inner membranes of the posterior acrosome display characteristic pentalaminar structure due to apposition of dense layers on both sides [27, 28]. The inner matrix has ladderlike densities [29, 30]. During the acrosome reaction, the rostral acrosomal content is dispersed by a vesiculation process of the outer acrosomal and plasma membranes. After the acrosome reaction and penetration through the zona pellucida, the posterior acrosome and the overlying plasma membrane remain intact [6]. The inner matrix of the posterior acrosome becomes narrower, but the ladderlike consistency appears more prominent [28, 30].
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In lower animals, sperm-oocyte fusion is initiated between the inner acrosomal membrane of the spermatozoan and the oocyte plasma membrane (reviewed in [31]). In mammals, the inner acrosomal membrane has developed into a resilient structure for effectively penetrating the egg vestments, albeit at the cost of fusibility, and the function of fusion has been taken up by the equatorial region. Conservation of the posterior acrosome segment in mammalian spermatozoa is perhaps an evolutionarily adapted strategy to ensure fusibility of the overlying equatorial plasma membrane [28]. The crucial role of the posterior acrosome during fertilization has been shown by the fact that antibody MN9, acting against its resident molecule equatorin, blocks sperm-oocyte fusion in vitro [21] and in vivo (unpublished communication with K. Yoshinaga et al.). In the present work, we have shown that residual equatorin is incorporated into the oocyte after fusion, along with the posterior acrosomal fragment. Equatorin is recognizable in one of the proembryonic cells after the first cleavage, but is undetectable after the second cleavage.
MATERIALS AND METHODS
Antibodies and Animals
The mAbs MN9 and MN7 were produced by immunizing female BALB/c mice with cauda epididymal spermatozoa of CD1 mice. Antibody production, purification, and their characterization have been described previously [16, 20, 21, 32, 33]. For the present work, the purified antibodies were used at 40x dilution. Anti-
-tubulin antibody was purchased from a commercial source (Zymed, San Francisco, CA) and used at 100x dilution. The tetramethyl rhodamine isothiocyanate (TRITC)-conjugated and fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibodies were purchased from Biosource (Camarillo, CA) and used at 80x and 40x dilutions, respectively. Five-nanometer colloidal gold-conjugated anti-mouse antibody was supplied by BioCell (Cardiff, UK) and was used at 40x dilution.
Mice used in the present experiments were the ICR strain (purchased from SLC Japan SLC Inc., Hamamatsu, Japan); females were 9–13 wk old, males were 11–15 wk old.
Spermatozoa Incubation and Calcium Ionophore Treatment
Modified Krebs-Ringer bicarbonate medium [34] was used for spermatozoa capacitation, fertilization, and gamete and embryo manipulations throughout the study, and will be referred to as mKR medium. Gamete and embryo incubations were performed in a 37°C CO2 incubator with an atmosphere of 5% CO2 in air. To investigate changes in the frequency of MN9 labeling, spermatozoa were cultured for various incubation periods, up to 24 h in mKR medium supplemented with 5 mg/ml BSA overlaid with mineral oil in 10-ml bottles at a concentration of 1–2 x 106/ml. For calcium ionophore treatment and sonication, an incubation period of 2 h was chosen. Calcium ionophore A23187 (Sigma, St. Louis, MO; stock solution 1 mM in dimethyl sulfoxide (DMSO) and stored at -30°C) was added to the sperm suspension to make the final concentration 10 µM. In the experiments for studying the effect of protease inhibitors, a cocktail of serine protease inhibitors was added 5 min before Ca2+-ionophore treatment. It consisted of 1 µg/ml pepstatin A, 10 µg/ml soybean trypsin inhibitor, 4 µg/ml aprotinin, 50 µg/ml p-aminobenzimidine hydrochloride, 10 µg/ml antipain, 2 µg/ml leupeptin, and 100 µM phenylmethyl-sulfonyl fluoride. The spermatozoa were further incubated for 1 h in a CO2 incubator after ionophore addition.
In some experiments, spermatozoa were treated with 0.1% Triton, 1 M NaCl, 10 mM NaOH, and 5 mM dithiothreitol (DTT) for overnight at 4°C, or with 1% SDS for 5 min at room temperature. They were washed, coverslips were applied, and they were directly labeled with antibodies without fixation or other treatment.
Sonication of Spermatozoa
The spermatozoa were prepared in a similar fashion as they were for the Ca2+-ionophore experiment. They were centrifuged at 500 x g for 5 min, the supernatants were removed, and the pellets resuspended in mKR-HEPES (NaHCO3 of mKR replaced with 20 mM Hepes) without BSA. In some experiments, the resuspending medium contained protease inhibitors as described above. The suspensions were allowed to stand for 5 min in a 37°C incubator at room atmosphere. Mild sonication was performed with a Branson Sonifier 250 (Danbury, CT) using a tapered, 1/2-inch horn. The sonication parameters that were followed for >90% head-tail breakage were 10% duty cycle, 5 output amplitude control, 20 output meter scale, and 15 pulses. The sonicated spermatozoa were allowed to stand for 5 min and then were centrifuged at 800 x g for 5 min. The pellets were resuspended in smaller volumes of mKR-Hepes. Coverslips were applied to aliquots and they were fixed for immunofluorescent studies.
Sperm Capacitation and Conventional In Vitro Fertilization
Female mice were stimulated to superovulate by injecting 7.5 IU eCG (Sankyo Zoki Co., Tokyo, Japan) and 48 h after that, with 7.5 IU hCG (Teikoku Zoki Co., Tokyo, Japan). Oocytes were collected 14–18 h after hCG injection. Spermatozoa capacitation and in vitro fertilization (IVF) were performed in mKR-BSA medium following methods described previously [21, 33]. The fertilized oocytes were fixed at 2, 6, and 24 h after insemination for immunofluorescent studies, and after 2 h for electron microscopic studies.
To obtain 4-cell proembryos, the oocytes were fertilized in mKR-BSA by incubating them with inseminated spermatozoa for 2 h as described above. They were washed with Chatot-Ziomek-Bavister medium (CZB) [35] without glucose but supplemented with 5 mg/ml BSA, and transferred to 50-µl drops of the same medium under oil, equilibrated in advance in a 37°C CO2 incubator. They were further cultured for 46 h (a total of 48 h after insemination). With this method, about 90% of the oocytes became fertilized, all of which developed to the 4-cell stage.
Zona-Free IVF
The cumulus-oocyte (CO) complexes from superovulated females were collected in mKR-Hepes. The cumulus cells were removed by repeatedly pipetting the CO complex in 0.1 mg/ml hyaluronidase (Sigma) in mKR-Hepes. After washing thoroughly, the oocytes were transferred to acid Tyrode solution (137 mM NaCl, 2.7 mM KCl, 1.3 mM CaCl22H2O, 0.5 mM MgCl2, 0.3 mM Na2HPO47H2O, 0.1 mg/ml glucose, 4 mg/ml polyvinylpyrrolidone [PVP; Sigma] pH 2.5) for a brief period. They were quickly transferred to a fresh drop of mKR-Hepes with 5 mg/ml PVP (Mr 3.6 x 105) and pipetted repeatedly to remove the distended zona. The zona-free oocytes were passed through several fresh drops of mKR-Hepes, and finally incubated in 0.5-ml drops of mKR-BSA overlaid with mineral oil (Sigma) and equilibrated in a 37°C CO2 incubator. The oocytes were allowed to recover for 3 h, after which 2 h-capacitated spermatozoa were added to achieve a final concentration of 5–6 x 104 spermatozoa/ml. The fertilized oocytes were fixed 30 min after insemination for immunofluorescent labeling.
Intracytoplasmic Sperm Injection and Sperm Head Microinjection
Whole sperm microinjections and sperm head microinjections were performed according to the methods described in earlier works [36, 37] with some modifications. The oocyte manipulations and culture were performed in mKR-HEPES supplemented with 5 mg/ml PVP (Mr 3.6 x 105) and mKR supplemented with 5 mg/ml BSA, as described above. Microinjections were done on a Zeiss inverted microscope equipped with an Eppendorf micromanipulator (Eppendorf, Hamburg, Germany) supplemented with a PrimeTech Piezo device, PMM-150 FU (PrimeTech Ltd., Nakamukaihara, Japan). Spermatozoa and sperm head were injected by blunt-end pipettes with an outer diameter 7 µm. For injecting sperm heads, fresh caudal spermatozoa were mildly sonicated as described above in ice-cold NIM (134 mM KCl, 2.6 mM NaCl, 7.8 mM Na2HPO4, 1.4 mM KH2PO4 pH 7.2 [37]) without protease inhibitors. The suspensions were washed with NIM once and kept in ice. The sperm heads were microinjected within 1 h after isolation. The oocytes that had undergone intracytoplasmic sperm injection (ICSI) and sperm head injections were incubated in mKR-BSA for 30 min or 5 h, and then processed for immunofluorescent studies.
Immunofluorescent Labeling
The oocytes were cleaned from the cumulus cells and denuded from the zona pellucida as described above. Fixation was done with 2% paraformaldehyde in PBS-PVP (PBS containing 5 mg/ml PVP) for 20 min. The oocytes were washed with PBS-BSA (5 mg/ml) and sequentially incubated with 1% Triton (30 min at 37°C), 10% normal goat serum (NGS, 30 min), MN9 antibody (45 min), TRITC-conjugated anti-mouse antibody (45 min), Hoechst 33258 (Sigma; 5 µg/ml, 10 min), and mounted with a drop of Vectashield (Vector Laboratories, Burlingame, CA). In some experiments, the microtubules were also labeled simultaneously. For that, the fixed and permeabilized oocytes were sequentially incubated with 10% NGS (30 min), MN9 antibody (45 min), TRITC-conjugated anti-mouse antibody (45 min), anti--tubulin antibody (45 min), FITC-conjugated anti-mouse antibody (45 min), Hoechst 33258 (5 µg/ml, 10 min), and mounted with a drop of Vectashield. The immunofluorescent studies were performed with an Olympus BX50 epifluorescent microscope (Olympus Optical Co., Tokyo, Japan), images were acquired with a CoolSNAP CCD (Roper Scientific Inc., Tucson, AZ), and processed with Adobe Photoshop 4.0 (Adobe Systems Inc., Mountain View, CA). Some micrographs were reconstructed by merging images taken at two focal planes.
The spermatozoa were double-labeled to study the acrosome and equatorin simultaneously. They were attached on polylysine-coated coverslips, fixed with 2% paraformaldehyde (20 min), and permeabilized with 1% Triton (30 min). The coverslips were washed with PBS and sequentially incubated with 10% NGS (30 min), MN9 antibody (45 min), TRITC-conjugated anti-mouse antibody (45 min), FITC-peanut agglutinin (FPNA; Honen Corp., Tokyo, Japan; 25 µg/ml, 45 min), Hoechst 33258 (5 µg/ml, 10 min), and mounted with a drop of Vectashield. In some experiments, the acrosomes were labeled with anti-acrin1 antibody (MN7) instead of FPNA. For that, the fixed and permeabilized spermatozoa were sequentially incubated with 10% NGS (30 min), MN9 antibody (45 min), TRITC-conjugated anti-mouse antibody (45 min), MN7 antibody (45 min), FITC-conjugated anti-mouse antibody (45 min), Hoechst 33258 (10 min), and mounted with a drop of Vectashield.
Control immunofluorescent studies were performed by labeling the fixed and permeabilized spermatozoa labeled with TRITC-anti-mouse antibody alone.
Electron Microscopy
For conventional transmission electron microscopy studies, the oocytes were removed from the fertilization medium 2 h after insemination. The cumulus cells were removed as described above, and the oocytes were fixed with 2.5% glutaraldehyde in PBS for 1 h. They were labeled with Hoechst 33342 (Sigma; 5 µg/ml in PBS) for 5 min. Desirable oocytes showing appropriate stages of sperm incorporation were selected by observing them through an epifluorescent microscope. The selected oocytes were postfixed with OsO4, embedded individually in 1.5% agar, dehydrated through a graded ethanol series, perfused with propylene oxide and embedded in Epon 812 (TAAB Labs., Berkshire, UK). Thin sections were stained with uranyl acetate and lead citrate, and studied through a JEM 1200 EX electron microscope (JEOL, Tokyo, Japan) operating at 80 kV. The images acquired on standard films were digitized with a Pictrostat 400 NSE scanner (Fuji Film, Japan).
For immunogold electron microscopy of the oocytes, their cumulus cells and zona pellucida were removed as described above. They were fixed with 2% paraformaldehyde and 0.25% glutaraldehyde for 1 h; quenched with 1 mg/ml sodium borohydride (30 min); extracted with 1% Triton (30 min at 37°C); and sequentially incubated with 10% NGS (1 h), MN9 antibody (1 h), and 5-nm colloidal gold conjugated anti-mouse antibody (1 h). After thoroughly washing the unbound colloidal gold, the oocytes were further fixed with 2.5% glutaraldehyde followed by 1% OsO4, and processed for embedding in Epon 812 as described above. The thin sections were stained with uranyl acetate.
Ultrastructural localization of equatorin was investigated in Ca2+-ionophore-induced, acrosome-reacted, and spontaneously acrosome-reacted spermatozoa. Ca2+-ionophore treatment was done as described above. The spermatozoa were washed and fixed with 2% paraformaldehyde for 20 min and sequentially incubated with 10% NGS (1 h), MN9 antibody (40x dilution, 1 h), and 5-nm colloidal gold conjugated anti-mouse antibody (1 h). After centrifuging and resuspending in PBS twice, the pellets were further fixed with 2.5% glutaraldehyde followed by 1% OsO4. The pellets were preembedded in 1.5% agar and processed for embedding in Epon 812 following a standard protocol. A population of spontaneously acrosome-reacted spermatozoa was obtained by incubating cauda spermatozoa in mKR-BSA for 2 h as described above. The sperm suspensions were centrifuged, the medium was removed down to 2 ml, and mAb MN9 was added to make the final dilution 40x. Further processing was performed as for Ca2+-ionophore-treated spermatozoa. The thin sections were stained with uranyl acetate.
Control immunogold labeling was done with Ca2+-ionophore-treated spermatozoa. They were processed in a fashion similar to that described above but without MN9 antibody treatment.
RESULTS
MN9 Labels the Equatorial Region of Acrosome-Reacted Spermatozoa
The present study confirms our earlier findings that mAb MN9 labels the equatorial region of mouse spermatozoa [20]. It did not label the anterior acrosomal sac of the intact capacitated cauda epididymal spermatozoa. After a 2-h capacitation, 11% of the spermatozoa displayed positive labeling with MN9 (Table 1). The frequency of labeling increased with incubation duration in the capacitating medium (Table 1). Simultaneous labeling with antibodies MN9 and MN7 or FPNA revealed that MN9 labels only those spermatozoa that have lost their acrosome entirely (Fig. 2, A and B). MN9 labeling of acrosome-reacted spermatozoa was further supported by Ca2+-ionophore and sonication experiments. These treatments resulted acrosome exocytosis in most of the spermatozoa (Table 1), all of which displayed MN9 labeling (Fig. 2, C and D). In the control preparations, TRITC-conjugated anti-mouse antibody alone did not label any recognizable structure (data not shown).
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The observation of MN9 labeling in acrosome-reacted spermatozoa could be the result of enzymes being released from the acrosome proteolytically modifying the equatorial proteins, rendering them reactive to MN9 antibody. Alternatively, disruption of the acrosomal sac may be necessary for permeabilization of the equatorial region. To discriminate between these two possibilities, capacitated spermatozoa were sonicated or treated with Ca2+-ionophore in the presence or absence of protease inhibitors. Spermatozoa that have a disrupted acrosome by sonication or Ca2+-ionophore displayed MN9 labeling, regardless of whether or not the treatment media contained protease inhibitors. Sonically severed sperm heads with an intact acrosome did not label with MN9 (Fig. 2D).
MN9 labeling is well preserved in fixed spermatozoa after extraction with 1% Triton X-100 for 30 min, the method that was followed for the routine immunofluorescent preparations. The spermatozoa subjected to overnight extraction at 4°C with NaCl, NaOH, or DTT (Fig. 2, E–G) or Triton (data not shown) did not show any appreciable loss of MN9 labeling. However, treatment with 1% SDS for 5 min completely extracted the protein (data not shown).
Ultrastructural localization of equatorin was investigated in acrosome-reacted spermatozoa by preembedding in immunogold for electron microscopy. Ionophore caused extensive membrane damage in the acrosome-reacted spermatozoa. The plasma membrane of the head region was lost in all affected sperm, and the outer acrosomal membrane of the posterior acrosome was damaged at several places, exposing the inner matrix or the inner acrosomal membrane. Colloidal gold particles were observed on those areas (Fig. 2H). In some spontaneously acrosome-reacted spermatozoa, the plasma membrane was retained at the equatorial region on which immunogold particles were heavily deposited (Fig. 2I). In such membrane-intact spermatozoa, the posterior acrosomes were visibly undamaged except for an opening at the anterior edge due to the loss of the anterior acrosomal sacs. In the control preparations prepared by excluding MN9 antibody treatment, none of the sperm organelles displayed significant gold particle labeling (data not shown).
Equatorin Enters into Oocytes along with the Sperm Head after Fusion and Degeneratesafter the Second Cleavage
Spermatozoa undergo a natural acrosomal reaction during passage through the oocyte vestments. Spermatozoa that penetrated into the perivitelline space displayed similar MN9 labeling to induced or spontaneously acrosome-reacted spermatozoa (Fig. 3A).
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MN9 labeling of zona-free IVF oocytes The early stage of sperm incorporation was studied in zona-free IVF. The oocytes fixed at 30 min after insemination displayed multiple sperm incorporation, second polar body ejection, and early stages of sperm head decondensation. The sperm head shed equatorin as an irregular mass while it was still in the cortical region, but it remained adjacent to the decondensing chromatin mass (Fig. 3B). As the sperm heads decondense further and move deep into the cytoplasm, the irregular masses of equatorin drift away from the nuclei and break down into smaller fragments.
MN9 labeling of IVF oocytes Two hours after insemination of the CO complex, the sperm head completely penetrates the oocyte, although some parts of the tail remain in the perivitelline space. In most oocytes, the fertilization cones were easily visible as bulged regions where the sperm head localized. The oocytes were fully activated, showing second polar bodies connected by compact residual bodies.
The oocytes fixed at 2 h after insemination displayed punctate but variously sized MN9 labeled bodies associated with the decondensing sperm heads (Fig. 3C). At 6 h after insemination, the IVF oocytes were observed at the pronuclear migration stage. The male and female pronuclei were surrounded by newly polymerized microtubules. During this period, punctate equatorin bodies seem to dissociate from the male nuclei, break down into big and smaller fragments, and localize at a distance from the male pronuclei, possibly pushed away by newly polymerized perinuclear microtubules (Fig. 3D). At 24 h after insemination, the fertilized oocytes divided into 2-cell proembryos, one of which invariably inherited the residual equatorin (Fig. 3E). The punctate equatorin structures were distinctly compact and smaller than those of the undivided zygotes. The proembryos fixed 48 h after postinsemination were at the 4-cell stage. Equatorin was undetectable in the majority of them (Fig. 3F). Less than 5% of proembryos displayed inconspicuous MN9 bodies in one of the proembryonic cells (Table 2). The extra spermatozoa remaining in the perivitelline space at this stage retained equatorin (Fig. 3F).
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Mode of Equatorin Degeneration after ICSI Is Similar to That of IVF
The ICSI procedure overrides the zona penetration and membrane fusion events, and the spermatozoa are directly deposited into the oocyte cytoplasm. Therefore, the spermatozoa are exposed to the oocyte cytoplasm without prior structural and molecular modifications that naturally occur during zona penetration and membrane fusion processes.
The oocytes fixed at 30 min after ICSI or sperm head injection exhibited intact sperm head and MN9 labeling as in acrosome-reacted spermatozoa (Fig. 4A). Oocytes fixed 5 h after postmicroinjection were fully activated, showing second polar body ejection and sperm head decondensation. Equatorin revealed by MN9 labeling appeared as small, irregular bodies, and either remained associated with the nuclei or moved away from them (Fig. 4B). In some oocytes, they disintegrated into smaller fragments. There were no recognizable differences in equatorin choreography during oocyte fertilization by ICSI or sperm head microinjection. Similar to IVF proembryos, residual equatorin bodies were observed in one of the blastomeres of 2-cell ICSI proembryos (Table 2).
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Electron microscopy of IVF oocytes Incorporation of equatorin into the oocyte cytoplasm after fertilization was further verified by conventional and immunogold electron microscopic studies. Oocytes fixed at 2 h postinsemination possessed double membranous structures of the posterior acrosome at the vicinity of the decondensing sperm heads (Fig. 5A). The width of the space between the membranes was apparently narrower than what has been found in the equatorial region of epididymal or perivitelline spermatozoa [6, 30]. The ladder-like densities were visible in the membranous fragments incorporated into the oocytes (Fig. 5B).
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Immunogold electron microscopic studies have confirmed that the membranous fragments near the decondensing sperm heads are the remnants of the equatorial region containing equatorin. In the preembedding immunogold labeling of the oocytes fixed at 2 h postinsemination, the bilamellar nature of the equatorial region was not clearly discernible. The gold particles were located on and around the fragments (Fig. 6, A–C). Analysis of the consecutive serial sections showed that fine, fuzzy, fibrillar materials appear in some places of the residual posterior acrosome, adorned with gold particles. The granular aggregates and the perforatorium (subacrosomal cytoskeleton) remnants occurring at the vicinity of the decondensing sperm heads were not labeled with the gold particles (Fig. 6D).
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DISCUSSION
Equatorin is exposed after acrosome reaction that coincides with the functional maturity of the overlying equatorial plasma membrane to participate in fusion. After sperm-oocyte fusion, it is incorporated into the oocytes along with the posterior acrosome fragments. Disintegration of the residual equatorin/posterior acrosome in the proembryonic cells is a slow process continuing through the second cleavage, similar to those of the tail components or mitochondria (Fig. 7).
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Equatorin and Other Equatorial Proteins Compared
Some sperm head proteins are known to be proteolytically modified or spatially reorganized during sperm maturation and the acrosomal reaction, making the proteins competent to bind the oocyte surface receptors and execute sperm-oocyte fusion [24, 38–42]. Likewise, the mAb MN9 reactive protein, equatorin, was detectable by immunofluorescent cytological technique (immunocyto technique) only in acrosome-reacted spermatozoa. A possibility that a pool of new MN9-reactive epitopes appeared on the equatorin region due to the proteolytic reaction of the acrosomal enzymes is unlikely because the presence of protease inhibitors during induced acrosome reaction and sonication did not inhibit this process. The conventional whole-mount immunocytotechnique was unable to detect equatorin in intact spermatozoa due to the impervious nature of the equatorial region. The equatorin was not removed from the sperm head even after rigorous extraction treatments, suggesting that its association with the inner equatorial matrix region is very stable.
Several other putative fusogenic equatorial proteins discovered by the antibody screening method resemble equatorin. The proteins recognized by the mAbs M1 [23] and M29 [22] have similar molecular sizes as that of equatorin, and block sperm-oocyte fusion without affecting binding. They are not detectable in intact spermatozoa by the conventional immunocytotechnique, but appear in the equatorial region after the acrosome reaction. There are, however, few important differences among them. The ultrastructural localization of the M29 protein is not known yet, whereas M1 protein localizes on the plasma membrane overlying the equatorial region but not in the inner matrix of the posterior acrosome, where equatorin resides. Nevertheless, extrapolated speculations provided by the authors point out scopes for supplementary works before drawing any definite conclusion. For example, Noor and Moore [23] assumed that M1 protein originally localizes in the intracellular site of the intact spermatozoa and diffuses to the exterior plasma membrane after the acrosome reaction. For ascertaining the presence of M1 protein in the intracellular site (possibly in the internal matrix of the posterior acrosome), postembedding immunogold electron microscopy would be required. Due to the impervious nature of this region, the preembedding immunogold labeling electron microscopy technique employed by the authors would detect only the external antigens. On the other hand, our previous work has shown the ultrastructural localization of equatorin in the posterior acrosome of intact spermatozoa by postembedding immunogold electron microscopy [20]. The present observation of immunogold labeling in the disrupted posterior acrosomal regions of Ca2+-ionophore-treated spermatozoa supports the fact that equatorin is originally localized inside the posterior acrosome. Most acrosome-reacted spermatozoa induced by Ca2+-ionophore lacked plasma membrane in the head region. Spontaneously acrosome-reacted spermatozoa that retained plasma membrane in the equatorial region possessed a heavy deposition of gold particles there, as shown in Figure 2I. Evidently, some equatorin is released from the internal site and adsorbed on the overlying plasma membrane after acrosome disruption. Although these observations highlight our earlier expectations that equatorin may diffuse outside and associate with the equatorial plasma membrane rendering it fusible with the oocyte microvilli [21, 43], it has yet to be seen whether similar events occur in zona-penetrated spermatozoa. There are some other circumstantial evidences to support equatorial relocation of fusogenic proteins after acrosome reaction [24, 44–46].
Disintegration of Equatorin in the Oocyte Cytoplasm
Sperm-oocyte fusion is the pivotal event of fertilization in which the sperm equatorial region plays a crucial role. The inner and outer acrosomal membranes and the plasma membrane of the equatorial region remain intact after the complete acrosomal reaction and zona penetration [47]. The classical electron microscopic studies have shown convincingly that sperm-oocyte membrane fusion takes place at the sperm equatorial region [6, 30, 48], whereas the posterior acrosome itself is engulfed by the oocyte microvilli in a phagocytic manner [6, 48]. The present study has shown that the posterior acrosome and the associated equatorin enter deep into the oocyte cytoplasm along with the sperm head. Although the outer acrosomal membrane of the posterior acrosome closely apposes with the oocyte plasma membrane during the fusion process [30], it is not left behind on the oocyte surface. The equatorial segment and equatorin contained in it are reflected away from the nuclei, possibly due to chromatin swelling and nuclear membrane reconstruction. They remain at the vicinity of the sperm head for a considerable length of time during the first cell cycle and after that, are inherited by one of the proembryonic cells. After ICSI the equatorial segment is directly exposed to the oocyte cytoplasm without prior interaction with the cortical membrane system, but displays similar cellular events of equatorin degeneration as the oocytes after IVF. These observations argue in favor of membrane interaction not being a prerequisite for shedding the equatorial posterior acrosome, equatorin, and their subsequent disintegration after ICSI.
The persistence of equatorin through early proembryonic cleavages is comparable with that of sperm tail microtubules and the midpiece mitochondrial sheath. The residual tail microtubules are retained up to the 8-cell [8] or blastocyst stage [4], and the mitochondrial sheath in bovine zygotes, until the late 4-cell stage [9]. However, the residual equatorin seems to degenerate a little early, before the 4-cell stage. The rationale of the moderate durability of equatorin in proembryos could be related to its stability against the effect of chaotropic agents such as NaCl, NaOH, Triton, or DTT. Although some portions of the membranous structure of the posterior acrosome seem to degenerate during the early stage, the residual equatorin did not disperse substantially. Possibly, it is eventually removed from the embryonic cells through the lysosomal pathway [49], similar to the sperm-borne mitochondria [11, 12].
An interesting question is, What could be the function of equatorin in the oocyte? Although some equatorin may be redistributed to the equatorial plasma membrane and participate in membrane fusion (discussed above), the significance of the majority that incorporates into the oocyte is largely ambiguous. Because its dispersal in the oocyte cytoplasm does not precede the second polar body ejection, cortical granule exocytosis, or pronuclear decondensation, the oocyte activation is less likely to be provoked by this process. Earlier, this function was believed to be played by a 30-kDa equatorial protein, oscillin [25, 50]. But some recent findings indicate that the oocyte-activating sperm factor may be a different protein [51–53]. We do not know whether a subpopulation of equatorin disperses during the fusion process and activates the oocytes.
Equatorin Degeneration in the Oocyte Cytoplasm Differs from the Degeneration of Other Sperm Head Components
As may be expected, the fate of equatorin during fertilization is remarkably different from that of other proteins that are associated with the sperm head surface. Among several such proteins identified in a wide variety of mammalian spermatozoa (discussed above), the fate of some SNAREs has been studied during IVF and ICSI [26]. SNAREs occur in the acrosomal membranes of intact spermatozoa but become restricted to the equatorial region after the acrosome reaction. After sperm-oocyte fusion, unlike equatorin, the residual SNAREs are not detectable [26]; apparently, they are left behind in the fused membrane and are diffused thereafter. If SNAREs are introduced into an oocyte along with a spermatozoan by ICSI, their disaggregation from the sperm head and disintegration are substantially delayed [26, 54]. Another sperm head component being investigated is the perinuclear theca, a cytoskeletal structure consisting of several proteins located beneath the acrosome in the anterior region and in between the plasma membrane and nuclear membrane in the posterior region [55]. In the equatorial region, it lies beneath the narrow posterior acrosome, where equatorin resides (Fig. 1). The fate of the perinuclear theca during fertilization seems to be essentially similar to that of the membrane-associated proteins such as SNAREs. As shown in bovine IVF, the perinuclear theca is firmly associated with the sperm head during fusion and disappears soon after penetration [10]. But when spermatozoa are introduced by ICSI into bovine oocytes, it remains intact for a long time in the oocytes that fail to activate [10]. However, in activated rhesus monkey oocytes after ICSI, the residual perinuclear theca is seen around the anterior segment of the male pronuclei that lags behind in decondensation [54, 56], similar to SNAREs during ICSI [26, 54]. It is worthwhile to note that the antibody used in the perinuclear theca studies was a polyclonal antibody, and that it reacted with several proteins and various structures of the sperm head [10], including the outer layer of the posterior equatorial segment [10, 55]. Therefore, it is not unlikely that some components of the perinuclear theca revealed by the polyclonal antibody are removed during sperm penetration. Moreover, in the present electron microscopic study, some dense materials were found to accompany the equatorial membranous structures incorporated into the oocytes after fertilization. It remains to be seen whether they are the perinuclear theca component. Because equatorin lies outside the perinuclear theca domain and is physically separated from the nuclei by a membrane barrier, its lesser tenacity with the nuclei during fertilization is not unexpected.
ACKNOWLEDGMENTS
We are thankful to Ms. H. Kiyotake for art works and to Dr. D.K. Saxena for various help.
FOOTNOTES
First decision: 30 April 2001.
1 K.T. is a recipient of a Grant-in-Aid for Scientific Research (12670022) from the Ministry of Education, Science, Sports and Culture of Japan. 
2 Correspondence: K. Toshimori, Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Kihara 5200, Miyazaki 8891692, Japan. FAX: 81 985 85 1363; ktoshi{at}post.miyazaki-med.ac.jp
Accepted: June 27, 2001.
Received: March 29, 2001.
REFERENCES
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