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Testis |
a Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Miyazaki 889-1692, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, fertilization, oocyte development, ovum, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As crucial as fertilization is, extensive studies over the past decade have shown that the molecular machinery associated with sperm-borne oocyte activating complex remains a mystery. Earlier studies have shown that the oocyte-activating complex of spermatozoa is a soluble cytosolic protein [9], it is cross-reactive across phylogenetically distant species [10–12], and may consist of one or more components [13, 14]. However, its molecular nature and subcellular localization are largely unknown. Earlier it was believed to be a 30- to 33-kDa protein, oscillin (Escherichia coli glucosamine 6-phosphate deaminase homolog) that is localized in the sperm head equatorial region [15]. These conclusions were subsequently refuted because recombinant oscillin, a protein without a known signaling domain [16], failed to activate oocytes upon microinjection [17]. In previous reports we have shown that the major sperm head equatorial protein, equatorin, is incorporated into oocytes after fertilization but it persists up to the 2-cell generation [18], arguing against its possible involvement in oocyte activation. Newly emerging evidence indicates that the sperm head perinuclear theca (PT) is involved in oocyte activation during fertilization [6, 19, 20]. Among the various peptides contained by the mammalian sperm head PT [21–23], some laboratories have focused on PT32, a possible initiator of the oocyte activation cascade [24]. In a preliminary report, PT32 was shown to be present in the postacrosomal region of the sperm head [24].
Perinuclear theca has a resilient cytoskeletal structure comprising various subdomains [22, 23, 25], each of which probably has a specialized and unique function. It is possible that the anterior PT located between the nuclear and inner acrosomal membranes mediates nuclear shaping during spermiogenesis, binds the acrosome to the nucleus until the acrosome reaction occurs [23, 26], and perforates the zona after the acrosome reaction [27]. The postacrosomal PT is generally believed to be involved in oocyte activation ([20, 28] and references therein). Some antibody probes that are known to react with the postacrosomal PT are antibodies to calicin [25], a 58-kDa polypeptide [29], the antithecin antibodies [22], and the MN13 antibody [30]. The anticalicin antibody reaction was observed in a hamster sperm extract that activates mouse oocytes upon microinjection [20]. Earlier we reported a monoclonal antibody (mAb), MN13, that labeled the postacrosomal region (PAR) of a wide variety of mammalian spermatozoa and recognized the paracrystalline structure of the postacrosomal PT [30]. In the present work we used the antibody to probe the postacrosomal PT and investigated its fate during fertilization and its role in oocyte activation. The findings provide evidence that it is likely to be involved in oocyte activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monoclonal antibody MN13 is an M type immunoglobulin. It was concentrated from a spent cell culture medium and kept at -70°C until used [30]. Monoclonal antibodies MN9 and MN7 are G type immunoglobulins, and were also purified from spent culture media [31, 32]. In the present study the antibodies were used at a 40:1 dilution. Anti-
-tubulin antibody was purchased from Zymed (San Francisco, CA) and used at a 100:1 dilution. Tetramethylrhodamine isothiocyanate (TRITC)-conjugated and fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibodies were purchased from Biosource (Camarillo, CA) and used at 80:1 and 40:1 dilutions, respectively.
The mice used in the present experiments were ICR and BDF1 strains, 9- to 13-wk-old females and 11- to 15-wk-old males purchased from SLC (Japan SLC Inc., Hamamatsu, Japan). Animal handling was performed in accordance with our institutional guidelines for the care and use of laboratory animals and with the approval of the Animal Research Committee of Miyazaki Medical College (approval 1998-001-5).
Calcium Ionophore Treatment and In Vitro Fertilization
Modified Krebs Ringer bicarbonate medium (mKR) [33] was used for spermatozoa capacitation, fertilization, and gamete and embryo manipulations. Gamete and embryo incubations were performed in a 37°C CO2 incubator in an atmosphere of 5% CO2 in air. Spermatozoa were incubated in the medium for 2 h prior to Ca2+ ionophore treatment and standard and zona-free in vitro fertilization (IVF) procedures. Detailed methods are described in an earlier paper [18].
For zona insemination, cumulus cells were removed by treatment with hyaluronidase (0.1 mg/ml in mKR-Hepes). The oocytes were inseminated for 20 min with spermatozoa that had been capacitated for 2 h, fixed, and processed for simultaneous labeling with MN7 and MN13 antibodies.
Zona-penetrated perivitelline spermatozoa were obtained by inseminating oocytes, the perivitelline spaces of which had been increased by removing some of the cytoplasm. After removal of cumulus cells, the oocytes were manipulated by following a procedure similar to that of intracytoplasmic sperm injection (ICSI). The oocytes were held with 80-µm-wide holding pipettes. Blunt-end ICSI needles 10 µm in diameter were inserted through the zona with the aid of a piezo drill from the opposite site of the metaphase II spindle area. Oocyte cytoplasm was aspirated without breaking the plasma membrane until 6- to 8-µm-wide perivitelline spaces were created. The needles were retracted, thereby stretching the thin cytoplasmic strands, which finally severed. The oocytes were incubated in IVF medium, inseminated with spermatozoa that had been capacitated for 2 h, and fixed 30 min later.
Spermatozoa Sonication and Extraction
Sonication and extractions were carried out in nucleus isolation medium (NIM) [20]. Mild sonication was performed at room temperature with a Sonifier 250 (Branson, Danbury, CT) using a tapered tip [18]. For dithiothreitol (DTT) extraction, the sonicated spermatozoa were resuspended in freshly prepared 15 mM DTT in NIM and incubated in a 26°C water bath for 30 min. After the treatment the suspension was centrifuged at 4°C and the pellet was resuspended in ice-cold NIM. Heat extraction was performed with NIM by incubating the sonicated spermatozoa suspension in a 45°C water bath for 30 min.
In experiments that involved protease inhibitors, a cocktail of protease inhibitors was added to the cold sonication medium. The cocktail 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. After sonication, the suspension was applied on coverslips, fixed, and labeled with antibodies.
Antibody Labeling of Sperm Heads for Microinjections
After DTT extraction the sperm heads were divided into three aliquots: 1) unlabeled, 2) labeled with MN9, and 3) labeled with MN13. Each aliquot was pelleted and resuspended in 40:1 diluted antibodies or blank NIM. Incubation was performed in a 4°C refrigerator for 1 h. After labeling, the suspensions were washed three times with ice-cold NIM.
Sperm Head Microinjection
Sperm head microinjections were performed with blunt-end pipettes using a PrimeTech piezo (PrimeTech Ltd., Ibaraki, Japan)[18]. Before ICSI, the sperm head suspensions were mixed with an equal amount of 20% polyvinyl pyrrolidine and were microinjected within 1 h after being mounted on the micromanipulator stage. Injected oocytes remained on the microscope stage (17°C) for 30 min. The degenerating oocytes were removed and the healthy ones were transferred to CZB-BSA that had been pre-equilibrated in a 37°C CO2 incubator, cultured for various times, and then processed for immunofluorescent studies.
Immunofluorescent Labeling
Sperm heads were double-labeled with MN7-MN13 or MN9-MN13 antibodies and the microinjected oocytes were double-labeled with MN9-antitubulin or MN13-antitubulin antibodies. The MN13 labeling of sperm heads and oocytes was detected with FITC-conjugated anti-mouse immunoglobulin (Ig) M antibody, whereas MN9 and MN7 labeling was revealed with TRITC-conjugated anti-mouse IgG antibody. Detailed methods of the immunofluorescent labeling have been described in a previous paper [18]. The zona-inseminated oocytes and zona-free IVF oocytes were comounted with sonicated sperm heads for comparative MN13 labeling. For control immunofluorescent studies, the DTT-extracted sperm heads were labeled with FITC-antimouse IgM or TRITC-antimouse IgG antibodies.
Images were acquired with a CoolSNAP charge-coupled device (Roper Scientific Inc., Tucson, AZ) and processed using Photoshop 6.0 software (Adobe Systems Inc., Mountain View, CA). Some micrographs were reconstructed by merging images obtained at two focal planes. Chi-square tests were performed using 2 x 2 contingency tables and the critical values of the chi-square distributions were derived from the standard table by using one degree of freedom.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monoclonal antibody MN13 labels the mouse sperm head postacrosomal PT [30] but the pattern of labeling was variable in intact spermatozoa. Some spermatozoa exhibited a thick, compact labeling, whereas others showed a rarefied and granular pattern (Fig. 1). Among freshly isolated epididymal spermatozoa, 8% displayed a positive MN13 reaction (Fig. 1A). The control preparations that were labeled only with FITC antimouse IgM antibody had no recognizable structure. The Ca2+ ionophore induced the acrosome reaction in about 85% of spermatozoa (unlabeled with MN7), but MN13 labeling was observed in only 11.5%, similar to that observed for spermatozoa that had been incubated for 2 h (11.2%; P > 0.2; Table 1).
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MN13 Epitope Is Expressed after Permeabilization of the Postacrosomal Region and Proteolytic Reaction by the Acrosomal Enzymes
Mild sonication resulted in head-midpiece breakage and equatorin exposure (labeling with MN9) in more than 90% [18] and acrosome loss in 67% of spermatozoa (Table 1). It is noteworthy that the proportion of the MN13 labeling increased to 71.3% after sonication (Table 1, Fig. 1B). All sperm heads displayed a uniformly labeled postacrosomal PT with MN13 after extraction with DTT or hot (45°C) NIM after sonication (Table 1, Fig. 1, C and D). The sonication and extraction procedures also resulted in an optimal exposure of MN9 labeling in the equatorial region (Fig. 1C). When protease inhibitors were added to the sonication medium, only 13% of spermatozoa exhibited MN13 labeling, and the frequency was not significantly different from that of 2 h incubated, untreated spermatozoa (P > 0.2; Table 1). The presence of protease inhibitors in the medium had no effect on the exposure of equatorin, which was labeled by MN9 antibody [18].
Spermatozoa Do Not Display MN13 Labeling During Zona Binding, Zona Penetration, or Fusion with the Oocyte
Spermatozoa undergo the physiological acrosome reaction while passing through the cumulus cells or after binding to the zona pellucida [27, 34]. All zona-attached spermatozoa were acrosome reacted because they did not label with anti-acrin1 antibody (mAb MN7). None displayed MN13 labeling (Fig. 2A), indicating that during fertilization the induced acrosome reaction does not lead to MN13p expression. Unfused perivitelline spermatozoa were observed in oocytes in which the cytoplasm had been partially removed. Such spermatozoa reacted with MN9 but showed no labeling with MN13 (Fig. 2B). Zona-free oocytes fixed at 30 min after insemination showed early stages of fertilization, possessing decondensing sperm heads with associated equatorin (Fig. 2C). Zygotes did not display detectable MN13 labeling.
Microinjection of DTT-Extracted, Unlabeled, or MN9-Labeled Sperm Heads Activates Metaphase II Oocytes
Mild sonication followed by DTT treatment permeabilizes the postacrosomal region, exposing equatorin and MN13p. The microinjection of DTT-extracted sperm heads activated 78.3% of metaphase II oocytes (Table 2). To estimate the activation rate, oocytes were examined 5 h after ICSI and were judged on the basis of observed immunofluorescent cytomorphologies after microtubule and DNA labeling. The activated oocytes showed a complete absence of metaphase II spindles and they possessed male and female pronuclei, but the polar bodies were often lost during immunofluorescent processing. Inactive oocytes displayed distinct metaphase plates and spindles. The microinjection of MN9 labeled sperm heads activated oocytes at a lower frequency (70%) than the unlabeled sperm heads (78.3%), but the difference was not statistically significant as revealed by a chi-square test (P > 0.2). Hence, the oocyte activating factor of sperm heads is not affected by MN9 labeling. Nevertheless, with MN9-labeled sperm head microinjections, higher proportions of pathological developments were observed such as fragmentation (Fig. 4D), loss of male chromatin instead of pronuclei formation, and some oocytes that showed fragmented cleavages (Table 2).
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Oocytes microinjected with DTT-extracted sperm heads and labeled with MN9 antibody revealed punctate equatorin bodies during pronuclear decondensation (5 h after injection; Fig. 3A) and in 2-cell pre-embryos (24 h after injection; Fig. 3B).
Microinjection of MN13-Labeled Sperm Heads Activates Oocytes at a Lower Rate
The microinjection of MN13-labeled sperm heads activated 37.9% of the oocytes. Normal male and female pronuclei were observed in 10.3% of the cases, whereas the majority of the activated oocytes (15.5%) developed fragmented male chromatin, although their female pronuclei appeared normal (Table 2). About 7% of the oocytes displayed only female pronuclei; the male chromatins probably were lost by fragmentation (Table 2). About 62% of oocytes failed to activate after MN13-labeled sperm head injection (Table 2). Most (53.4%) possessed intact metaphase II spindles and sperm PCC invested by metaphase-like spindles. Some oocytes (8.6%) possessed fragmented or deformed sperm heads without microtubular vestment. The oocytes remained in the metaphase II stage with sperm head PCCs even after 24 h of incubation, suggesting a persistent failure to activate (Fig. 4I). Other anomalies in some oocytes included randomly dispersed chromatin, accompanied by splitting of the spindles or absence of spindle around the sperm head PCCs.
Application of chi-square tests showed highly significant differences between the rates of oocyte activation after MN13 labeled/unlabeled sperm head microinjections (0.0001 < P < 0.001) and MN13/MN9 labeled sperm head microinjections (0.0001 < P < 0.001). These calculations are consistent with the hypothesis that MN13 labeling of sperm heads diminishes their oocyte activating function.
MN13p Is Transiently Relocated to the Spindle Polar Regions Before It Disappears
Microinjected oocytes fixed after 30 min of recovery at 17°C displayed intact sperm heads in the cytoplasm. However, in most cases, the putative MN13p appeared to dissociate from the postacrosomal PT region of sperm heads (Fig. 4, A and B). Upon further incubations, the redistribution of MN13p was notably different in activated oocytes compared with inactive oocytes. When incubated at 37°C for an additional 30 min to 2 h after microinjection, the activated oocytes exhibited early stages of pronuclear decondensation, and midzone spindles between the second polar bodies and female pronuclei. Both pronuclei were completely devoid of MN13 labeling and the labeling was redistributed on the midzone spindle, mostly in the distal (polar) regions (Fig. 4, G and H). In inactivated oocytes, the dissociated MN13 labeled granules were redistributed around the metaphase II and the PCC spindle poles (Fig. 4, D and E). Various transitional stages of MN13p migration from the decondensing male pronuclei or sperm heads to the spindle poles were observed in different oocytes (Fig. 4, C and D). At least 5 h after microinjection incubation, MN13-labeled structures were completely lost from activated oocytes or were rarely detectable in the spindle polar regions of inactive oocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the presence of protease inhibitors, the MN13 epitope remains unexpressed even after sonication, suggesting that the antibody recognizes the proteolytically modified peptide. In intact spermatozoa the plasma membrane overlying the postacrosomal region protects the internal matrix from exogenous proteases and restricts the penetration of antibodies. The postacrosomal region plasma membrane remains intact even after acrosome exocytosis or zona penetration [35], thus preventing acrosome-reacted spermatozoa from labeling with MN13 antibody. For the same reasons, the fertilizing ability of spermatozoa is not affected by the presence of the antibody in the IVF medium (our unpublished observation). These observations further indicate that postacrosomal region constituents are preserved after passing through the oocyte vestments and are delivered intact to oocytes after fusion. Some spermatozoa that expose MN13p may be dead or unable to activate oocytes if they are introduced via ICSI [36].
MN13p remains undetectable even after sperm incorporation during zona-free IVF. Because the nascent postacrosomal PT was unreactive to MN13, its fate during fertilization was investigated by extracting sonicated sperm heads with DTT in order to expose their MN13 epitope, followed by their microinjection into oocytes. Microinjected sperm heads directly interact with oocyte cytoplasm without prior structural or molecular modifications that occur during zona penetration and the membrane fusion events of natural fertilization. Nevertheless, the birth of normal siblings after ICSI in several mammalian species including mice [20] is consistent with the normality of the ICSI process. After sperm head ICSI, the MN13-labeled bodies are dispersed in the oocyte cytoplasm during the early stages of fertilization after sperm head ICSI and are transiently relocated to the spindle polar regions (Fig. 4, D–F), resembling the distribution of centrosomal proteins in an acentriolar mouse oocyte [37, 38]. However, a centrosomal function of MN13p is less likely because it moves to the spindle's polar region of the pre-existing metaphase II spindles but it does not nucleate them. Microtubule nucleation was not observed from the residual MN13 bodies in the other regions of oocyte cytoplasm. Several proteins are known to accumulate in the spindle polar regions [39], possibly as the result of a dynein-like motor activity [40]. In activated oocytes the residual MN13p adsorbed on the midzone spindles are destined to be discarded into the perivitelline space after completion of the second polar body cytokinesis. It is worthwhile to mention that several key players of cell cycle regulation are also localized in the spindle polar regions [41–43].
Some other PT proteins appear to behave differently than MN13p during fertilization. For example, calicin remains associated with the sperm head postacrosomal region during fusion and early stages of chromatin decondensation [44]. A component of the PT that is resorbed into the oocyte cortex during IVF [19] may be retained intact if the spermatozoa are deposited deep into the oocyte by ICSI [45]. However, in mice, the MN13-reactive postacrosomal PT appears to be less tenaciously associated and disperse more rapidly than previously studied PT components. In comparison, the equatorin of microinjected sperm heads was detectable up to the 2-cell stage, as was found in IVF pre-embryos [18]. It should be noted that MN13p was exposed as a result of proteolytic enzyme activity, and hence might behave differently than the nascent peptide. Nevertheless, to understand the precise role of postacrosomal PT during fertilization, it is necessary to generate an antibody against the nascent epitope that labels the residual postacrosomal PT of spermatozoa in the oocyte cytoplasm after in vivo or in vitro fertilization.
Among various domains of the sperm head, the postacrosomal region deserves special attention because of its possible involvement in oocyte activation [20, 28]. During fertilization, the postacrosomal PT is exposed to the oocyte cytoplasm before any other region of the sperm head. The anterior sperm head along with the anterior PT is engulfed in a phagocytic manner and is enclosed by the reverted oocyte plasma membrane [46, 47], and as a result, would slowly interact with the oocyte cytoplasm. The most probable sperm head component that might trigger the oocyte activation cascade is MN13p of the postacrosomal PT. Its localization on the outermost layer of the postacrosomal PT [30] is ideal for interacting with the oocyte cytoplasm immediately after sperm-oocyte membrane fusion, in contrast to those that are localized in the inner layers [25, 29]. To obtain conclusive evidence, it would be necessary to isolate the putative protein, determine its sequence, verify the signaling domain, microinject it into oocytes, and examine the resulting activation.
A further complication arises in the antigenic epitope exposure because the MN13 reactive band at 40–60 kDa was vanishingly weak in immunoblots [30]. We have yet to devise an alternative method for isolating MN13p. Nevertheless, the postacrosomal PT or MN13p can be exposed by sonication and DTT extraction, and can be reacted with the antibody, thus neutralizing its function. The microinjection of sonicated and DTT-extracted sperm heads activates oocytes (also see [13, 14]), whereas after MN13 labeling, sperm heads failed to activate oocytes to a significant extent, providing indirect evidence that MN13p is involved in oocyte activation. It should be noted that some of the oocytes were still activated by MN13-reacted sperm heads, but most displayed pathological features.
Based on the above findings, it can be assumed that MN13 binding neutralizes a portion of the total oocyte-activating factor exposed in the postacrosomal PT due to sonication and DTT extraction. The unexposed anterior PT that is unreactive to MN13 may harbor additional oocyte activating factors. Most of the oocytes activated by MN13-reacted sperm head microinjection showed male chromatin fragmentation, suggesting that the reduced dose of oocyte activating factor of such sperm heads is not sufficient to induce male pronuclear decondensation. Differential effects of microinjected oocyte activating factors on various events of activation have been pointed out in some earlier studies [14, 48]. It could be further argued that sonicated sperm heads might lose oocyte-activating factor during incubation with the antibody or the antibody itself might disable the signal transduction process by unspecific binding. These possibilities are less likely because the sperm heads prepared in a similar manner but labeled with anti-equatorin antibody MN9 activated oocytes to the same extent as unlabeled sperm heads. Moreover, sperm head manipulation and incubation at low temperature do not involve any noticeable loss of oocyte activation factor [13].
The oocyte activating factor (referred to as the sperm-borne oocyte activating factor—SOAF—by Kimura et al. [20]) has been shown to be comprised of a soluble-heat labile and insoluble-heat resistant components [13, 14]. The soluble component can be extracted with 15 mM DTT, whereas the heat-stable component remains firmly associated with the sperm heads even after the removal of all soluble components by hot medium (45°C) treatment. None of them alone can activate oocytes, but when they were coinjected they elicited full activation [13]. The present study indicates that MN13p probably corresponds to the insoluble, heat-stable component of the SOAF because the antibody labels sperm heads optimally after being extracted with 45°C NIM. Thus far, the molecular characterization of SOAF or the biochemical pathway of its involvement in oocyte activation has not been proposed, but evidence exists to suggest that the proteolytic reaction is necessary for the generation of the soluble component [14]. In this light, the possibility cannot be ruled out that the MN13p may be a residual peptide remaining after the soluble component is proteolytically cleaved and removed. The epitopes for antibodies PN-1 and PN-2 are probably generated in this way in the postacrosomal PT [22]. These comparisons are largely speculative and, moreover, the proteolytic processing of the remaining SOAF may be executed by endogenous proteases [14], whereas the generation of the epitope recognized by MN13 is due to an exogenous protease reaction that does not occur during natural fertilization. Even though MN13p is formed by proteolytic modification of the nascent postacrosomal PT after sonication and DTT extraction, its oocyte activating function is unaffected.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: K. Toshimori, Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Kihara 5200, Miyazaki 889-1692, Japan. FAX: 81 985 85 1363; ktoshi{at}post.miyazaki-med.ac.jp
3 Current address: S141, Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65211
Received: 1 April 2002.
First decision: 19 April 2002.
Accepted: 9 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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