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Gamete Biology |
Center for Research for Reproduction and Women's Health,3 Department of Gynecology, University of Pennsylvania Medical Center, BRB II/III, Philadelphia, Pennsylvania 19104
Department of Anatomy,4 Miyazaki Medical College, Kiyotake, Miyazaki 889-1653, Japan
Department of Anatomy and Developmental Biology,5 Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 260-8670, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, in vitro fertilization, male reproductive tract, sperm, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Earlier reports suggest that basigin is involved in multiple processes such as spermatogenesis [14], implantation [15], and sperm-oocyte interactions [16]. These reports are based on gene knockout mouse models or studies using polyclonal anti-basigin antibodies. Unlike anti-basigin antibody, anti-MC31 monoclonal antibody (mMC31) specifically recognizes MC31 molecules on the flagella of rat sperm [17]. Thus, mMC31 could be a useful tool to study the molecular dynamics and functional aspects of this molecule during events of fertilization.
Although the change of MC31 during epididymal maturation has been reported earlier [8], the molecular nature of the MC31 molecule and its physiological role during fertilization remain largely unknown. Although MC31 is believed to be a glycoprotein, to our knowledge, no experimental evidence has been published to demonstrate the nature and extent of glycosylation of MC31 in cauda and capacitated spermatozoa.
In the present study, we examined the glycosylation status and molecular changes of MC31 during capacitation and the acrosome reaction because glycosylation is important for the structural stability and function of other glycoproteins [18]. We also studied the possible role of MC31 during the fertilization process using mMC31. Finally, we present here a proposed model incorporating the predicted structure of MC31/CE9 correlated with its processing and suggest that the changes observed in MC31 are physiologically significant.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wistar rats of 3 wk (female) and 12 wk (male) in age were obtained commercially (Kyudo Company, Kumamoto, Japan) and maintained thereafter in the laboratory animal center under the animal welfare guidelines of our college. The animals were anesthetized with diethylether and then killed by cervical dislocation.
Reagents, In Vitro Fertilization (IVF), Culture Medium, and Antibodies
All chemicals used in this study were of analytical and culture grade and obtained from Nacalai Tesque Inc. (Kyoto, Japan) unless otherwise stated. A modified Krebs-Ringer bicarbonate solution [19] was used with slight alterations: HEPES was not added, the concentration of BSA was increased to 6 mg/ml, and the pH of the media was adjusted to 7.4 for gamete manipulation and fertilization. We refer to this media as rat fertilization medium (RFM) throughout this study. The protein (antibody) concentration was estimated by the bicinchoninic acid method according to the supplier's manual (Pierce Chemical Co., Rockford, IL).
Purified monoclonal antibody (mAb) mMC31 (IgG) was used as a probe in this study [17]. Another mAb, mMC71 (IgG) that does not cross-react with rat spermatozoa, [20] and preimmune rat serum were used as controls.
Antibodies mMC31and mMC71 were purified by a previously described method [20] using a Protein A column (Ampure PA kit; Amersham Biosciences, Little Chalfont, UK) and desalted with a RFM medium pre-equilibrated Sephadex G 25M column (PD-10 column) to exchange binding buffer with RFM medium. The resulting purified antibody (2.0–3.7 mg/ml peak yield) was stored at -80°C until used for the IVF study.
Sperm Collection, Capacitation, and Induction of Acrosome Reaction
Spermatozoa were collected from the cauda epididymidis of mature male rats. The distal portion of each epididymis was cut using a blade, and a dense sperm mass was squeezed out of the epididymis. The spermatozoa were allowed to disperse into 400 µl RFM medium under oil in an IVF dish. The spermatozoa were diluted in paraformaldehyde (PFA) (1:500) and counted using a hemocytometer. The final concentration of spermatozoa was adjusted to 2 x 106 spermatozoa/ml in RFM medium, and these spermatozoa were incubated for 5 h at 37°C in a 5% CO2 environment for capacitation [21]. The capacitated spermatozoa were coincubated with 0.1 mM progesterone for 1 h to induce an acrosome reaction in the presence or absence of mMC31.
Determination of Molecular Mass by Western Blotting
Testicular, epididymal, and capacitated sperm samples were extracted with 0.1% Triton X-100 in 0.1 M phosphate buffered saline (PBS) containing several proteinase inhibitors: 0.2 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO), 1 mg/ml pepstatin A, 1 mg/ml leupeptin (Nacalai Tesque, Kyoto, Japan), and were shaken for 30 min at 0°C. After centrifugation at 20 000 x g for 30 min, the supernatant was precipitated with ice-cold acetone. The extract was dissolved in a sample buffer, which consisted of 1% SDS, 1 mM phenylmethylsulfonyl fluoride, 20% glycerol, 62.5 mM Tris-HCl, and bromophenol blue. The samples were then analyzed by SDS-PAGE according to the procedure of Laemmli [22] using a 10% polyacrylamide gel. Western blotting was done according to the method of Towbin et al. [23]. The blot was immunostained using the anti-MC31 antibody as the primary antibody and horseradish peroxidase-conjugated anti-rabbit IgG (Protos Immunoresearch, Burlingame, CA) as the secondary antibody. The molecular mass was estimated by visual estimation, referring to standard high/low molecular markers (Sigma) and plotting on a semilogarithmic paper to estimate the relative distances (molecular mass).
Deglycosylation of MC31
The deglycosylation of extracted proteins from cauda and capacitated spermatozoa was performed using a deglycosylation kit (Prozyme Inc., San Leandro, CA) according to the manufacturer's recommendation. In brief, MC31 extracts were prepared as described above and treated with peptide-N-glycosidase (PN glycosidase) for 24 h. Changes in molecular mass were determined by SDS-page and Western blotting as described above.
Localization of MC31 on Capacitated and Acrosome-Reacted Spermatozoa
Spermatozoa that completed a 5-h incubation in capacitation media, in which most of the spermatozoa showed a whiplash movement (hyperactivation), were referred to as capacitated spermatozoa, while spermatozoa from cauda epididymidis before incubation were referred to as uncapacitated spermatozoa (control). Isolated cauda epididymal and capacitated spermatozoa were fixed in 1% paraformaldehyde for 20 min and washed three times with 0.1 M PBS by centrifugation at 450 x g for 5 min. Spermatozoa were incubated with mMC31 antibody for 1 h and washed again. Thereafter, these spermatozoa were incubated with secondary antibody, a goat fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG + IgM (Biosource International, Camarillo, CA) for 30 min. The washing procedure was repeated again and the samples were placed on glass slides. Immunofluorescence images and the corresponding differential interference contrast (DIC) images of the slides were observed using a fluorescence microscope (BX-50 type; Olympus, Tokyo, Japan).
Immunocytochemical Localization of MC31 under IVF Conditions (IVF-Indirect Immunofluoresence [IIF] Assay)
Immunocytochemical localization of MC31 on living capacitated and acrosome-reacted spermatozoa under IVF conditions was performed as previously described [24]. Living spermatozoa were immunostained before fixation, and then the motility was reduced by adding 0.1% sodium azide (finally less than 0.1%). During this treatment, spermatozoa still showing very slow movement were referred to as living spermatozoa and were subjected to assessment of the localization of MC31. In brief, spermatozoa were capacitated in RFM medium at 37°C for 5 h and then mMC31 (at 100 µg/ml concentration) was added to the capacitation medium. After incubation for an additional 1 h, these spermatozoa were fixed in 1% PFA for 20 min and washed three times with 0.1 M PBS by centrifugation at 450 x g for 5 min. The resulting spermatozoa were incubated for 30 min with FITC-conjugated goat anti-mouse IgG + IgM as the secondary antibody.
Immunocytochemistry of Oocyte
Immunostaining of the oocytes was performed in the same way as described above to determine if the mMC31 antibody cross-reacted with oocyte components.
Assessment of Acrosomal Status (Acrosome Reaction)
To assess the status of the acrosome, sperm samples were washed three times with 0.1 M PBS and double labeled with anti-MC31 antibody and mMN7 as primary antibodies using routine IIF techniques as described above using mouse FITC-conjugated goat anti-mouse IgG + IgM and IgG (H+L) rhodamine (TAGO Inc., Burlingame, CA) as secondary antibody. The monoclonal antibody mMN7 recognizes an intra-acrosomal antigen molecule, acrin 1, in rat, mouse, and human [24–26]. Images were obtained using the green channel, the red channel, and differential interference contrast using Coolsnap digital video-PC system (Roper Scientific, Trenton, NJ) equipped with light microscope (BX-50 type). Images were further processed with Adobe Photoshop software (Adobe System Inc., Mountain View, CA), and printed in a Pictrostat digital 400 printer (Fuji Film, Tokyo, Japan). At least 300 spermatozoa were counted.
In Vitro Fertilization Experiments
To assess the effect of the mMC31 antibody on fertilization, female rats (3 wk old) were superovulated by consecutive intraperitoneal injections of eCG 15 IU and 48 h later with hCG 20 IU. Ovulated egg masses were collected from oviducts at 20 h after hCG administration. The oocyte-cumulus complexes were released by rupturing the oviductal ampullae and immediately transferred into the RFM, which had been equilibrated with 5% CO2 in air under oil (dimethyl polysiloxane; Sigma).
Preparation of the Zona-Free Oocytes
The oocyte-cumulus complexes were briefly treated with 0.01% hyaluronidase in RFM to remove the cumulus masses. The zona pellucida-free oocytes were prepared by briefly incubating the cumulus-free oocytes with low-pH (pH 2.5) RFM medium (modified Krebs-Ringer bicarbonate solution) [19], as described elsewhere in the case of mice with low pH (pH 2.5) TYH medium [24].
The zona pellucida-intact oocytes were inseminated with capacitated sperm in RFM containing mMC31 antibody at 0.0 (control), 50, or 100 µg/ml. Acrosome-reacted spermatozoa, which were treated either in the presence of mMC31 antibody or in RFM only, were also used to inseminate zona pellucida-free oocytes in RFM containing mMC31 antibody at 0.0 (control) or 100 µg/ml. The final concentration of spermatozoa was adjusted to approximately 4 x 105 spermatozoa/ml in a 400-µl drop of RFM.
Assessment of IVF Events
The percentage of motile spermatozoa was estimated by the hanging drop preparation method [27]. Sperm motility and fertilization events, such as pronucleus formation and two-cell formation in RFM, were monitored on an inverted microscope (IM type; Olympus) through a charge-coupled device camera (Model KP-M1; Hitachi Denshi Ltd., Tokyo, Japan) at 30 min, 1 h, 5 h, 20 h, and 40 h after insemination.
Fertilization
Fertilization was determined by phase-contrast microscopy. The embryo showing pronucleus formation, at 8 h or more, or two cells at 24 h after the coincubation of gametes were considered fertilized.
Statistical Analysis
Each experiment was repeated three to five times. The data were analyzed using the Student t-test (Table 1) and chi-square test (Table 2). Data were considered statistically significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As evident from the immunoblot (Fig. 1), mMC31 antibody recognizes three bands between 28 and 26 kDa in caudae epididymides. A slight modification was observed after capacitation. Capacitated spermatozoa showed two major bands of 28 and 26 kDa.
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Treatment with PN glycosidase treatment reduced the molecular mass of MC31 from three bands in the range of 28–26 kDa to a single band of 23–20 kDa in cauda and capacitated spermatozoa (Fig. 1), providing strong evidence that MC31 is glycosylated.
Changes in Localization of MC31 on Spermatozoa During Epididymal Maturation (IIF)
Immunostaining of spermatozoa from capita, corpora, and caudae epididymides revealed that MC31 is localized in the principal piece of testicular and caput spermatozoa. During epididymal transit, MC31 gradually relocates toward the midpiece. In the corpus epididymis, spermatozoa showed uniform staining on the whole tail (PP+MP), but MC31 was present predominantly on the midpiece of spermatozoa retrieved from the cauda epididymidis (Table 1).
Changes in Localization of MC31 on Spermatozoa During Capacitation and Acrosome Reaction (the Modified IIF Assay, First Antibody Incubation under IVF Condition: IVF-IIF)
On approximately 70% capacitated spermatozoa (fixed in PFA), MC31 was predominantly localized on the midpiece of the sperm tail (MP in Table 1), exhibiting stronger staining compared with cauda spermatozoa. Besides the predominant localization of MC31 on the midpiece, various other staining patterns were found; these included staining on the sperm head, on the middle piece only, or on both the regions (Fig. 2). Several spermatozoa showed no staining, representing a negative sperm population. Only 1.5% of the fixed cauda spermatozoa showed head staining, but the percentage of head staining was higher (approximately 23%) when capacitated, unfixed spermatozoa were immunostained under IVF conditions (Table 1). Other data summarizing various staining patterns are also listed in Table 1. Prolonged incubation under capacitation conditions increased the percentage of head staining. A maximum of 40% sperm-head labeling was found after 24 h of incubation (data not shown). Some spermatozoa also showed staining on the equatorial segment. When the acrosomal status was determined by tracing the immunocytochemistry images for the intra-acrosomal antigen, acrin 1 (MN7), the sperm showing equatorial segment staining with mMC31 were not immunostained with the pertinent antiacrin 1 antibody, mMN7 (not shown); the absence of acrin 1 staining indicates that spermatozoa are acrosome reacted. The percentage of spermatozoa showing equatorial segment staining increased to 25% after the progesterone-induced acrosome reaction (Fig. 3, a–c).
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Effect of mMC31 Antibody on In Vitro Fertilization Events Sperm Motility
The mMC31 antibody had no effect on sperm motility. The average percentage of motile spermatozoa was 67.5% ± 7.0% in the presence of mMC31 antibody as compared with controls in RFM only and in the presence of mMC71 (74.0% ± 9.0% and 66.5% ± 3.7%, respectively).
Fertilization Rate in the Zona Pellucida-Intact Experiment
Pronucleus formation was suppressed in a dose-dependent manner at 20 h after coincubation of the gametes in the presence of the mMC31 antibody. The fertilization rate was only 35.2% ± 3.4% in the presence of mMC31 antibody at 100-µg/ml concentration, but 90% in control groups and above 80% in mMC71 (83.2% ± 7.3%) (Table 2). Similarly, two-cell embryo formation was only 40.7% ± 7.0% in the mMC31 antibody at 100-µg/ml concentration, but above 90% in control groups (Table 2, and Fig. 4). The difference between the experimental groups and the control groups was statistically significant (P < 0.001).
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Fertilization of the Zona Pellucida-Free Oocytes
Spermatozoa were able to bind to zona pellucida-free oocytes equally well in both the experimental group treated with mMC31 antibody and the control groups (RFM only and RFM plus mMC71). However, the percentage of fertilization was significantly lower (P < 0.001) in the experimental group treated with mMC31 at 100-µg/ml concentration (28.5% ± 5.1%) compared with control groups, RFM only (78.2% ± 7.4%) and RFM plus mMC71 (73.4% ± 9.5%) (Table 3). Similarly, two-cell embryo formation was significantly lower (P < 0.001) in the experimental groups (mMC31) when compared with the control groups (RFM and RFM plus mMC31) as shown in Table 3.
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Proposed Model of MC31
The polypeptide backbone MC31/CE9 consists of 272 amino acids (Accession No. CAA47655) [1, 28]. This molecule consists of a signal peptide of 21 amino acids and mature peptide of 251 amino acids. Structurally, the amino acid sequence encodes two Ig domains.
Immunostaining of Oocyte with mMC31 Antibody
Because preincubation of gametes with mMC31 inhibited fertilization (Tables 2 and 3), oocytes were treated with antibody to determine if mMC31 cross-reacts. Because the antibody did not cross-react with any component of oocytes (not shown), we conclude that the inhibitory action of mMC31 on fertilization is a direct effect on spermatozoa, not on the oocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We also present a model structure of MC31/CE9 and domain architecture of Ig domains retrieved by conserved domain architecture retrieval tool (Fig. 5). The Ig domain of MC31/CE9 exhibited a higher degree of homology with 1MCP H (monocyte-chemoattractant protein-1), 1ZXQ (intercellular adhesion molecules ICAM-2), and many other molecules [5, 29]. ICAM-2-like molecules act as ligand for integrin. Recognition by integrin proteins on the cell surface regulates the adhesive interactions between cells and their surroundings. MC31/CE9 may be involved in sperm-oocyte interactions.
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Structural analysis of MC31 indicates that MC31/CE9 of testicular sperm contains two Ig domains. The first Ig domain (toward the N-terminus) is removed as spermatozoa mature in the epididymis. This leaves a single Ig domain that may be involved in homologous oligomerization. Two putative N-glycosylation sites remain in this structure.
We further examined if the mature MC31 molecule is glycosylated and the molecular changes (if any) taking place during capacitation are the consequence of deglycosylation or protein processing. Treatment of cauda and capacitated sperm extracts with PN glycosidase caused a reduction in the molecular mass (23–20 kDa), approximating the predicted size for the mature polypeptide alone (19 332 Dalton) (Fig. 1). These findings suggest that cauda MC31 is a glycosylated molecule with N-linked oligosaccharides. In addition, use of the NetOGlyc 2.0 server (http://www.cbs.dtu.dk/services/NetOGlyc/) predicts that threonine-150 (found in the second, V-type Ig domain) of the mature glycoprotein may contain O-linked glycans.
Although removal of sugars by the deglycosylation kit used in this study led to a product similar in size to that predicted for the protein moiety, other posttranslational modification may occur. For example, protein motif analysis predicts protein kinase A phosphorylation sites in the cytoplasmic domain of MC31/CE9. The finding that MC31/CE9 is initially localized in the principal piece of the flagellum is interesting because the major protein of this structure is AKAP4, a protein that anchors protein kinase A [30], suggesting that the C-terminal tail of MC31/CE9 could be a target for regulation by phosphorylation. Further studies should focus on this possibility and examine whether phosphorylation of MC31/CE9 during capacitation is related to the redistribution of this protein from the flagellum to the head.
The molecular mass of MC31/CE9 did not show a remarkable change after capacitation, suggesting that neither major deglycosylation nor protein processing takes place during capacitation. However, such changes during the acrosome reaction cannot be ruled out. Although capacitation did not cause a further reduction in the size of MC31, only two prominent bands were clearly visible after capacitation, as compared with three distinct bands in the case of cauda spermatozoa, suggesting some molecular modification might have occurred during capacitation (Fig. 1).
Sperm maturation and capacitation may not only cause changes in the molecular mass but also alter the localization of MC31. As reported earlier, MC31 was found to shift from the principal piece to the midpiece during epididymal maturation [17]. The present finding demonstrates a further shift in its localization to the sperm head after capacitation and to the equatorial segment after the acrosome reaction (Figs. 2 and 3, Table 1). This phenomenon of change in localization might be related to the molecular migration or an unmasking of the epitope recognized by mMC31 during capacitation and the acrosome reaction. The relocation of sperm surface molecules during capacitation and the acrosome reaction have been reported earlier in cases of certain other sperm molecules, 2B1 [31, 32] and PH20 [33], as well as CE9 [28] and mouse basigin [16]. After the acrosome reaction, the PH-20 surface antigen of guinea pig spermatozoa migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane [8]. Our findings with MC31 resemble that of 2B1, which also reportedly appears on the sperm head after a prolonged incubation [31, 32].
After capacitation and the acrosome reaction, MC31 appears on the sperm head and at the equatorial segment (ES), respectively (Figs. 2 and 3, Table 1). The monoclonal antibody mMC31 also significantly inhibited fertilization of zona pellucida-intact oocytes. This inhibition might result from the poor adhesion of the sperm to the zona pellucida. However, in the case of rat fertilization, it is difficult to quantify the sperm binding to the zona pellucida because spermatozoa penetrate the zona soon after binding. Our earlier study has shown that anti-basigin antibody against the homologous protein in mouse inhibits primary sperm binding [16].
Fertilization of zona pellucida-free oocytes was also inhibited significantly in the presence of mMC31. This suggests involvement of migrated or newly exposed MC31 molecules on the sperm head after acrosome reaction in the sperm-oocyte fusion process. The fact that MC31 found on the ES and acrosomal antigen, acrin 1, is not present in the same sperm when traced using mMN7 antibody confirms that the sperm has completed the acrosome reaction. In general, acrin 1 is dispersed during the acrosome reaction and does not remain on a completely reacted sperm [24]. Also, mMC31 antibody does not cross-react with the oocyte (data not shown).
The mechanism of sperm-oolemma interactions presently remains unclear. This step is thought to be mediated by several molecules, like MN9, M29, DE, and several others, on the sperm head, specifically on the ES (see the review by McLeskey et al. [34]). It is possible that MC31 might act as an agonist or an antagonist to some sperm and/or oocyte molecules, which directly facilitate sperm-oocyte fusion.
Contact between a spermatozoon and an oocyte plasma membrane stimulates a series of interactions, primarily involving integrin/disintegrin binding [33, 35]. Other surface glycoproteins, including major histocompatibility complex (MHC) products [36] and surface molecules, which have structural homology with MHC-class antigens such as EMMPRIN/bsg, may influence sperm-oolemma recognition, binding, and/or fusion.
Several transmembrane glycoprotein molecules have been reported to be integrin-associated, such as EMMPRIN, basigin, HT7, or CE9 [37]. MC31 is an integrin-associated molecule. Oocyte integrins such as 
6/ß1 serve as receptors for the complementary sperm molecules involved in sperm-oocyte binding or fusion. Thus, it is also possible that anti-MC31 antibodies might affect the interaction of these molecules, consequently inhibiting fertilization. Recognition by integrin protein on the cell surface regulates the adhesive interactions between cells and their surroundings. MC31 exhibits structural similarity with ICMA-1, 2, and 3. The intercellular adhesion molecules ICMA-1, 2, and 3 are like VCAM-1 [29] members of the immunoglobulin superfamily that are recognized by an I domain-containing integrin, lymphocyte function associated antigen 1 (LFA-1, or CD1 a/CD18).
In summary, MC31 appears to facilitate sperm-oocyte interaction. It is likely that a migrated or newly exposed MC31 molecule, on the equatorial segment of acrosome-reacted spermatozoa, directly facilitates the sperm-oocyte interaction, or alternatively, acts in combination with other molecules. Further study will be needed to reveal the exact molecular mechanism of its action during fertilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: K. Toshimori, Department of Anatomy and Developmental Biology (G1), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 260-8670, Japan. FAX: +81 43 226 2019; ktoshi{at}faculty.chiba-u.jp
Received: 31 July 2003.
First decision: 21 August 2003.
Accepted: 25 November 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6ß1 functions as a sperm receptor. Cell 1995 81:1095-1104[CrossRef][Medline]3ß1 complexes with emmprin/basigin/OX47/M6. J Biol Chem 1997 272:29174-29180This article has been cited by other articles:
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  M. S. Wakle, S. A. Joshi, and V. V. Khole Monoclonal Antibody From Vasectomized Mouse Identifies a Conserved Testis-Specific Antigen TSA70 J Androl, November 1, 2005; 26(6): 761 - 771. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
