MOLECULAR REPRODUCTION AND DEVELOPMENT 29:289-293 (1991)
A Monoclonal Antibody, MN13, That Recognizes Specifically a Novel Substance Between the Postacrosomal Sheath and the Overlying Plasma Membrane in the Mammalian Sperm Head K. TOSHIMORI,' I. TANII,' C. OURA' AND E.M. EDDY2 'Department of Anatomy, Miyazaki Medical College, Miyazaki, Japan; 2Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina ABSTRACT Monoclonal antibody MN13 raised against mouse spermatozoa specifically recognizes the postacrosomal region of the sperm head in several mammalian species. Colloidal gold-immunoelectron microscopy of demembranated mouse spermatozoa indicated that the antigen is associated with the outer layer of the periodic substructure apparently linking the postacrosomal sheath to the overlying plasma membrane. The antigen recognized by MN13 may contribute to the intimate association of the postacrosomal sheath with the overlying plasma membrane.
Substances located between the postacrosomal sheath and the overlying plasma membrane have not been identified previously. In this study, we report that monoclonal antibody (MAb) MN13 specifically recognizes a substance present in this location.
MATERIALS AND METHODS MAb Production MAb MN13 (L8F3/2D10)was produced by immunizing female BALB/c mice with washed CD-1 cauda epididymal spermatozoa, and antibody-producing cell lines were selected by procedures described previously Key Words: Postacrosomal region, Cytoskeleton, UI(Fenderson et al., 1984).MAb MN13 was determined to trastructure, lmmunocytochemistry be a subtype M immunoglobulin (Ig), using the mouse monoclonal subisotyping kit 55050-K (Hyclone Laboratories, Logan, UT). The method was a highly sensiINTRODUCTION tive enzyme immunoassay using rabbit antimouse Ig The postacrosomal sheath is a subcellular structure performed according to the supplier's protocol. The of the sperm head extending from the posterior margin spent culture medium containing MN13 was concenof the acrosome (equatorial segment) anteriorly to the trated approximately 40 times using Minitan filtration posterior ring distally and interposed between the system with molecular weight cut of 30,000 (Millipore outer nuclear membrane and the plasma membrane Corporation, Bedford, MA) and stored at -80°C until (Fawcett, 1975) (Fig. 1). The postacrosomal sheath is an use. electron-dense layer 10-15 nm thick. It is connected to the cytoplasmic face of the plasma membrane by periIndirect Immunofluorescence (IIF) and odic substructures that emerge from the postacrosomal Avidin-Biotin Peroxidase Complex (ABC) sheath. These periodic substructures form what has Immunohistochemistry been called the paracrystalline sheet (Longo et al.?1987) The methods used were described previously (Toshior the paracrystalline substructure (Olson and Winfrey, mori et al., 1988, 1990). 1988). The periodic substructure could affect the properties of the overlying plasma membrane because of Immunogold Staining for Electron this intimate association. It is of functional significance Microscopy (IGS-EM) that the portion of the plasma membrane that is CD-1 mouse spermatozoa from the cauda epididymiadherent to the postacrosomal sheath is involved in the dis were washed twice in phosphate-buffered saline bitial stage of sperm-egg fusion (Yanagimachi, 1988; (PBS) containing protease inhibitors; 1 mM phenylOura and Toshimori, 1990). methylsulfonyl fluoride (PMSF; Sigma Chemical Co., Previous studies have identified a 60,000 dalton (60 St Louis, MO), 10 p.g/ml soybean trypsin inhibitor (STI; kD) protein, termed calicin (Longo et al., 19871, and a 58 kD protein in the postacrosomal region of bull spermatozoa (Olson and Winfrey, 1988). Immunocy- Received January 14, 1991; accepted February 22, 1991. tochemical methods were used to show that these Address reprint requests to Kiyotaka Toshimori, MD, Department of proteins are located in the space between the postac- Anatomy, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, rosomal sheath and the underlying nuclear membrane. Japan. 0 1991 WILEY-LISS, INC.
290
K. TOSHIMORI ET AL.
Sigma), and 100 d m l Aprotinin (Sigma) (PBSI). Thereafter, the washed spermatozoa were demembranated with 0.1% Triton X-100 in PBSI for 30 rnin on ice. After being washed three times in PBSI, the spermatozoa were treated with PBS containing 1%normal goat serum (PBS/NGS) for 30 min on ice. The treated spermatozoa were then centrifuged at approximately 2,OOOg for 2 min, and the pellet was suspended in MAb MN13 diluted to 1/40 in PBS/NGS at room temperature (RT) for 60 min. Then the spermatozoa were washed in PBS containing 0.1%bovine serum albumin (PBSIBSA) three times at approximately 200g for 5 min. The sperm pellet was then suspended and incubated with colloidal gold particles (15 nm) conjugated with antimouse IgM (Janssen Pharmaceuticals, Beerse, Belgium) diluted 1/20in PBS/BSA at RT for 60 min. The treated samples were washed in PBS/BSA three times for 10 min each. The washed samples were fixed with 2.5% glutaraldehyde in PBS for 1hr, washed, dehydrated with a graded ethanol series, and embedded in Epon 812. Some samples were treated with tannic acid as described previously (Toshimori et al., 1985). Ultrathin sections were cut with a glass knife on an LKB Ultratome and mounted on copper grids. Sections were stained with uranyl acetate and lead citrate and examined in a JEOL 200CX electron microscope with a goniometer stage and operated at 80 kV accelerating voltage.
RESULTS Using ABC immunohistochemistry, it was found that MAb MN13 did not cross-react with somatic cells from mouse brain, heart, thyroid, pancreas, liver, ovary and uterus (data not shown). The schematic drawing shown in Figure 1 summarizes the organization and distribution of domains and membrane systems of the mammalian (mouse) sperm head. IIF studies showed that MAb MN13 binding was restricted to the postacrosomal region of the sperm head of mouse, rat, hamster, and human; the same domain was immunostained in all species examined (Fig. 2). Staining was constant and uniform when spermatozoa were treated mildly with a nonionic detergent such as 0.1% Tritin X-100 to remove the plasma membrane (demembranated sperm) (Fig. 21, but live spermatozoa were never stained. IGS-EM of demembranated mouse spermatozoa clearly demonstrated that immunogold particles were located exclusively on the outer surface of the periodic substructure of the postacrosomal sheath (Fig. 3a). As in IIF studies, this was seen only with sperm demembranated before MAb MN13 was applied. IIF and pre- or postembedding IGS-EM did not stain the nucleus-facing surface of the postacrosomal sheath, which was expected to be partially released from the nucleus by mechanical treatment such as more than 2 rnin ultrasonication at 80 W (50% cycle) using a Branson Sonifier 250 (Branson Ultrasonics, Danbury, CT). The electron-dense periodic substructures projecting from the postacrosomal sheath to the plasma mem-
Fig. 1. Schematic drawing of the mammalian (mouse) sperm head region. a: Longitudinal section, showing the various regions of the head. b Sagittal section of the head, showing the constituent membranes of each region. Abbreviations used in this figure and subsequent figures: A, acrosome; AA, anterior acrosome; ES, equatorial segment; IAM, inner acrosome membrane; INM, inner leaflet of nuclear membrane; IPM, inner leaflet of plasma membrane; N, nucleus; OAM, outer acrosome membrane; ONM, outer leaflet of nuclear membrane; OPM, outer leaflet of plasma membrane; PA, posterior acrosome; PAR, postacrosome region; PAS, postacrosomal sheath; PM, plasma membrane; PR, posterior ring; PS, periodic structure.
brane are well visualized in thin sections of tannic acid-treated spermatozoa. These periodic substructures appears to link the postacrosomal sheath to the overlying plasma membrane (Fig. 3b). DISCUSSION In this report we have demonstrated distribution of an antigen recognized by MAb MN13 that is restricted to spermatozoa. It is present between the postacrosomal sheath and the overlying plasma membrane and does not appear to correspond to any sperm component characterized previously. The periodic substructure of the postacrosomal sheath (paracrystalline substructure or paracrystalline sheet) is thought to be a sperm cytoskeletal component because of its resistance to extraction with detergent and high salt buffer and to be involved in attachment of the postacrosomal sheath to overlying plasma membrane (Longo et al., 1987; Olson and Winfrey, 1988). The antigenic substance recognized by MAb MN13 was associated with the outer surface of the periodic substructure after the overlying plasma membrane was removed with mild detergent treatment. This suggests that the antigenic substance may be involved in the attachment of the postacrosomal sheath to the overlying plasma membrane. A schematic illustration of this hypothesis is shown in Figure 4.
POSTACROSOMAL SHEATH STRUCTURE
291
Fig. 2. Light microscopy of several mammalian spermatozoon. a-d Phase-contrast image of spermatozoon. a’-d’: Indirect immunofluorescence with MAb MN13, showing that the antigen is restricted to the postacrosomal region of the same spermatozoan head. a, Mouse; b, rat; c, hamster; d, human. x 2,500.
It is unknown at present whether the antigenic substance of MAb MN13 is present on the nucleusfacing surface of the postacrosomal sheath. However, this seems unlikely in that immunohisto- and immunocytochemical staining such as IIF and pre- or postembedding IGS-EM in combination with mechanical treatment causing partial release of the postacrosomal sheath from the nucleus always failed to stain this region. These staining approaches could reflect, at least partially, the antigenic substance between the postacrosomal sheath and the underlying nucleus membrane. This remains to be assessed more rigorously.
Two proteins of 58 kD (Olson and Winfrey, 1988) and 60 kD (calicin) (Long et al., 1987) have been reported previously to be associated with the postacrosomal sheath. However, they are present between the postacrosomal sheath and the underlying nuclear membrane and are different from the antigenic substance recognized by MAb MN13. The molecular weight of the antigenic substance recognized by MAb MN13 remains to be determined. When the IIF method was applied, 1%sodium dodecyl sulfate completely abolished the antigenicity, but immunostaining of Western blots of spermatozoa extracts
292
K. TOSHIMORI ET AL.
Fig. 3. Electron microscopy of mouse spermatozoon. a: Immunoelectron microscopy utilizing MAb MN13-immunogold staining. Immunogold particles (arrows) are associated with the outer surface of the electron-dense periodic substructure emerging from the postacrosomal sheath. The plasma membrane formerly attached to the
postacrosomal sheath was removed with 0.1% Triton X-100 treatment prior to application of MAb MN13. x 159,000. b: Tannic acid-treated mouse spermatozoon. Electron-dense substructure (paracrystalline substructure)projects from the postacrosomal sheath to the overlying plasma membrane (arrows with asterisks). x 312,000.
,O P M I PM PS t t T t t T T T t t t t t t t t t* * * * * * * * * * * * * * * * -P A S
denaturing, or the antigen may be difficult to detect by this method because ofits low concentration relative to other sperm proteins. MAb MN13 specifically recognizes the postacrosomal region of spermatozoa of severa1 different mammals. Therefore, the antigenic sub-. stance appears to be well conserved among mammalian species.
I
x x x x x x x x x x x x x x x x - n N M- ..... 7 ,
-N Fig. 4. Schematic drawing demonstrating the hypothesis of the relationship of the postacrosomal sheath-associated substance recognized by MAb MN13. Small asterisks show the predominant localization of 58 kD protein reported by Olson and Winfrey (1988). Large asterisks show the distribution of the 60 kD protein reported by Longo et al. (1987). Solid circles on the outer surface of the periodic substructure between the postacrosomal sheath and the overlying plasma membrane show the predominant localization of the antigenic substance recognized by MAb MN13 reported in this study.
with MAb MN13 showed only weak staining of 40-60 kD component in the and an 48 kD component in the rat (unpublished data). The antigenic determinant (epitope) may be sensitive to
ACKNOWLEDGMENTS The authors thank M ~y. . Goto and y. ~ ~ jfori i assistance and Miss H. Kiyotheir excellent take for word processing. This study was supported by the Japanese Ministry of Education, Science and Culture (grant-in-aid for general scientific research N ~ . 62570011 to K.T. and 01570014 to c . 0 . and K.T. and grant-in-aid for encouragement of young scientist No. 01770021 to I.T.). REFERENCES Fawcett DW (1975):The mammalian spermatozoon. Dev Biol44:394436. Fenderson BA, O’Brien DA, Millette CF, Eddy EM (1984): Stage-
POSTACROSOMAL SHEATH STRUCTURE specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dev Biol 103:117-128. Longo FJ, Krohne G, Franke WW (1987): Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: A novel class of cyloskeletal elements. J Cell Biol 105:1105-1120. Olson GE, Winfrey, VP (1988): Characterization of postacrosomal sheath of bovine spermatozoa. Gamete Res 20:329-342. Oura C, Toshimori K (1990): Ultrastructural studies on the fertilization of mammalian ggmetes. Int Rev Cytol 122:105-151. Toshimori K, Araki S, Oura C (1988): Masking of sperm maturation
293
antigen by sialic acid in the epididymis of the mouse: An immunohistochemical study. Histochemistry 90: 195-200. Toshimori K, Araki S, Oura C (1990): Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface. Biol Reprod 42:151-160. Toshimori K, Higashi R, Oura C (1985): Distribution of intramembranous particles and filipin-sterol complexes in mouse sperm membranes: Polyene antibiotic filipin treatment. Am J Anat 174:455-470. Yanagimachi R (1988):Mammalian fertilization. In E Knobil, J Neill, et al. (eds): “The Physiology of Reproduction.” New York: Raven Press, pp 135-185.
A Monoclonal Antibody, MN13, That Recognizes Specifically a Novel Substance Between the Postacrosomal Sheath and the Overlying Plasma Membrane in the Mammalian Sperm Head K. TOSHIMORI,' I. TANII,' C. OURA' AND E.M. EDDY2 'Department of Anatomy, Miyazaki Medical College, Miyazaki, Japan; 2Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina ABSTRACT Monoclonal antibody MN13 raised against mouse spermatozoa specifically recognizes the postacrosomal region of the sperm head in several mammalian species. Colloidal gold-immunoelectron microscopy of demembranated mouse spermatozoa indicated that the antigen is associated with the outer layer of the periodic substructure apparently linking the postacrosomal sheath to the overlying plasma membrane. The antigen recognized by MN13 may contribute to the intimate association of the postacrosomal sheath with the overlying plasma membrane.
Substances located between the postacrosomal sheath and the overlying plasma membrane have not been identified previously. In this study, we report that monoclonal antibody (MAb) MN13 specifically recognizes a substance present in this location.
MATERIALS AND METHODS MAb Production MAb MN13 (L8F3/2D10)was produced by immunizing female BALB/c mice with washed CD-1 cauda epididymal spermatozoa, and antibody-producing cell lines were selected by procedures described previously Key Words: Postacrosomal region, Cytoskeleton, UI(Fenderson et al., 1984).MAb MN13 was determined to trastructure, lmmunocytochemistry be a subtype M immunoglobulin (Ig), using the mouse monoclonal subisotyping kit 55050-K (Hyclone Laboratories, Logan, UT). The method was a highly sensiINTRODUCTION tive enzyme immunoassay using rabbit antimouse Ig The postacrosomal sheath is a subcellular structure performed according to the supplier's protocol. The of the sperm head extending from the posterior margin spent culture medium containing MN13 was concenof the acrosome (equatorial segment) anteriorly to the trated approximately 40 times using Minitan filtration posterior ring distally and interposed between the system with molecular weight cut of 30,000 (Millipore outer nuclear membrane and the plasma membrane Corporation, Bedford, MA) and stored at -80°C until (Fawcett, 1975) (Fig. 1). The postacrosomal sheath is an use. electron-dense layer 10-15 nm thick. It is connected to the cytoplasmic face of the plasma membrane by periIndirect Immunofluorescence (IIF) and odic substructures that emerge from the postacrosomal Avidin-Biotin Peroxidase Complex (ABC) sheath. These periodic substructures form what has Immunohistochemistry been called the paracrystalline sheet (Longo et al.?1987) The methods used were described previously (Toshior the paracrystalline substructure (Olson and Winfrey, mori et al., 1988, 1990). 1988). The periodic substructure could affect the properties of the overlying plasma membrane because of Immunogold Staining for Electron this intimate association. It is of functional significance Microscopy (IGS-EM) that the portion of the plasma membrane that is CD-1 mouse spermatozoa from the cauda epididymiadherent to the postacrosomal sheath is involved in the dis were washed twice in phosphate-buffered saline bitial stage of sperm-egg fusion (Yanagimachi, 1988; (PBS) containing protease inhibitors; 1 mM phenylOura and Toshimori, 1990). methylsulfonyl fluoride (PMSF; Sigma Chemical Co., Previous studies have identified a 60,000 dalton (60 St Louis, MO), 10 p.g/ml soybean trypsin inhibitor (STI; kD) protein, termed calicin (Longo et al., 19871, and a 58 kD protein in the postacrosomal region of bull spermatozoa (Olson and Winfrey, 1988). Immunocy- Received January 14, 1991; accepted February 22, 1991. tochemical methods were used to show that these Address reprint requests to Kiyotaka Toshimori, MD, Department of proteins are located in the space between the postac- Anatomy, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, rosomal sheath and the underlying nuclear membrane. Japan. 0 1991 WILEY-LISS, INC.
290
K. TOSHIMORI ET AL.
Sigma), and 100 d m l Aprotinin (Sigma) (PBSI). Thereafter, the washed spermatozoa were demembranated with 0.1% Triton X-100 in PBSI for 30 rnin on ice. After being washed three times in PBSI, the spermatozoa were treated with PBS containing 1%normal goat serum (PBS/NGS) for 30 min on ice. The treated spermatozoa were then centrifuged at approximately 2,OOOg for 2 min, and the pellet was suspended in MAb MN13 diluted to 1/40 in PBS/NGS at room temperature (RT) for 60 min. Then the spermatozoa were washed in PBS containing 0.1%bovine serum albumin (PBSIBSA) three times at approximately 200g for 5 min. The sperm pellet was then suspended and incubated with colloidal gold particles (15 nm) conjugated with antimouse IgM (Janssen Pharmaceuticals, Beerse, Belgium) diluted 1/20in PBS/BSA at RT for 60 min. The treated samples were washed in PBS/BSA three times for 10 min each. The washed samples were fixed with 2.5% glutaraldehyde in PBS for 1hr, washed, dehydrated with a graded ethanol series, and embedded in Epon 812. Some samples were treated with tannic acid as described previously (Toshimori et al., 1985). Ultrathin sections were cut with a glass knife on an LKB Ultratome and mounted on copper grids. Sections were stained with uranyl acetate and lead citrate and examined in a JEOL 200CX electron microscope with a goniometer stage and operated at 80 kV accelerating voltage.
RESULTS Using ABC immunohistochemistry, it was found that MAb MN13 did not cross-react with somatic cells from mouse brain, heart, thyroid, pancreas, liver, ovary and uterus (data not shown). The schematic drawing shown in Figure 1 summarizes the organization and distribution of domains and membrane systems of the mammalian (mouse) sperm head. IIF studies showed that MAb MN13 binding was restricted to the postacrosomal region of the sperm head of mouse, rat, hamster, and human; the same domain was immunostained in all species examined (Fig. 2). Staining was constant and uniform when spermatozoa were treated mildly with a nonionic detergent such as 0.1% Tritin X-100 to remove the plasma membrane (demembranated sperm) (Fig. 21, but live spermatozoa were never stained. IGS-EM of demembranated mouse spermatozoa clearly demonstrated that immunogold particles were located exclusively on the outer surface of the periodic substructure of the postacrosomal sheath (Fig. 3a). As in IIF studies, this was seen only with sperm demembranated before MAb MN13 was applied. IIF and pre- or postembedding IGS-EM did not stain the nucleus-facing surface of the postacrosomal sheath, which was expected to be partially released from the nucleus by mechanical treatment such as more than 2 rnin ultrasonication at 80 W (50% cycle) using a Branson Sonifier 250 (Branson Ultrasonics, Danbury, CT). The electron-dense periodic substructures projecting from the postacrosomal sheath to the plasma mem-
Fig. 1. Schematic drawing of the mammalian (mouse) sperm head region. a: Longitudinal section, showing the various regions of the head. b Sagittal section of the head, showing the constituent membranes of each region. Abbreviations used in this figure and subsequent figures: A, acrosome; AA, anterior acrosome; ES, equatorial segment; IAM, inner acrosome membrane; INM, inner leaflet of nuclear membrane; IPM, inner leaflet of plasma membrane; N, nucleus; OAM, outer acrosome membrane; ONM, outer leaflet of nuclear membrane; OPM, outer leaflet of plasma membrane; PA, posterior acrosome; PAR, postacrosome region; PAS, postacrosomal sheath; PM, plasma membrane; PR, posterior ring; PS, periodic structure.
brane are well visualized in thin sections of tannic acid-treated spermatozoa. These periodic substructures appears to link the postacrosomal sheath to the overlying plasma membrane (Fig. 3b). DISCUSSION In this report we have demonstrated distribution of an antigen recognized by MAb MN13 that is restricted to spermatozoa. It is present between the postacrosomal sheath and the overlying plasma membrane and does not appear to correspond to any sperm component characterized previously. The periodic substructure of the postacrosomal sheath (paracrystalline substructure or paracrystalline sheet) is thought to be a sperm cytoskeletal component because of its resistance to extraction with detergent and high salt buffer and to be involved in attachment of the postacrosomal sheath to overlying plasma membrane (Longo et al., 1987; Olson and Winfrey, 1988). The antigenic substance recognized by MAb MN13 was associated with the outer surface of the periodic substructure after the overlying plasma membrane was removed with mild detergent treatment. This suggests that the antigenic substance may be involved in the attachment of the postacrosomal sheath to the overlying plasma membrane. A schematic illustration of this hypothesis is shown in Figure 4.
POSTACROSOMAL SHEATH STRUCTURE
291
Fig. 2. Light microscopy of several mammalian spermatozoon. a-d Phase-contrast image of spermatozoon. a’-d’: Indirect immunofluorescence with MAb MN13, showing that the antigen is restricted to the postacrosomal region of the same spermatozoan head. a, Mouse; b, rat; c, hamster; d, human. x 2,500.
It is unknown at present whether the antigenic substance of MAb MN13 is present on the nucleusfacing surface of the postacrosomal sheath. However, this seems unlikely in that immunohisto- and immunocytochemical staining such as IIF and pre- or postembedding IGS-EM in combination with mechanical treatment causing partial release of the postacrosomal sheath from the nucleus always failed to stain this region. These staining approaches could reflect, at least partially, the antigenic substance between the postacrosomal sheath and the underlying nucleus membrane. This remains to be assessed more rigorously.
Two proteins of 58 kD (Olson and Winfrey, 1988) and 60 kD (calicin) (Long et al., 1987) have been reported previously to be associated with the postacrosomal sheath. However, they are present between the postacrosomal sheath and the underlying nuclear membrane and are different from the antigenic substance recognized by MAb MN13. The molecular weight of the antigenic substance recognized by MAb MN13 remains to be determined. When the IIF method was applied, 1%sodium dodecyl sulfate completely abolished the antigenicity, but immunostaining of Western blots of spermatozoa extracts
292
K. TOSHIMORI ET AL.
Fig. 3. Electron microscopy of mouse spermatozoon. a: Immunoelectron microscopy utilizing MAb MN13-immunogold staining. Immunogold particles (arrows) are associated with the outer surface of the electron-dense periodic substructure emerging from the postacrosomal sheath. The plasma membrane formerly attached to the
postacrosomal sheath was removed with 0.1% Triton X-100 treatment prior to application of MAb MN13. x 159,000. b: Tannic acid-treated mouse spermatozoon. Electron-dense substructure (paracrystalline substructure)projects from the postacrosomal sheath to the overlying plasma membrane (arrows with asterisks). x 312,000.
,O P M I PM PS t t T t t T T T t t t t t t t t t* * * * * * * * * * * * * * * * -P A S
denaturing, or the antigen may be difficult to detect by this method because ofits low concentration relative to other sperm proteins. MAb MN13 specifically recognizes the postacrosomal region of spermatozoa of severa1 different mammals. Therefore, the antigenic sub-. stance appears to be well conserved among mammalian species.
I
x x x x x x x x x x x x x x x x - n N M- ..... 7 ,
-N Fig. 4. Schematic drawing demonstrating the hypothesis of the relationship of the postacrosomal sheath-associated substance recognized by MAb MN13. Small asterisks show the predominant localization of 58 kD protein reported by Olson and Winfrey (1988). Large asterisks show the distribution of the 60 kD protein reported by Longo et al. (1987). Solid circles on the outer surface of the periodic substructure between the postacrosomal sheath and the overlying plasma membrane show the predominant localization of the antigenic substance recognized by MAb MN13 reported in this study.
with MAb MN13 showed only weak staining of 40-60 kD component in the and an 48 kD component in the rat (unpublished data). The antigenic determinant (epitope) may be sensitive to
ACKNOWLEDGMENTS The authors thank M ~y. . Goto and y. ~ ~ jfori i assistance and Miss H. Kiyotheir excellent take for word processing. This study was supported by the Japanese Ministry of Education, Science and Culture (grant-in-aid for general scientific research N ~ . 62570011 to K.T. and 01570014 to c . 0 . and K.T. and grant-in-aid for encouragement of young scientist No. 01770021 to I.T.). REFERENCES Fawcett DW (1975):The mammalian spermatozoon. Dev Biol44:394436. Fenderson BA, O’Brien DA, Millette CF, Eddy EM (1984): Stage-
POSTACROSOMAL SHEATH STRUCTURE specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dev Biol 103:117-128. Longo FJ, Krohne G, Franke WW (1987): Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: A novel class of cyloskeletal elements. J Cell Biol 105:1105-1120. Olson GE, Winfrey, VP (1988): Characterization of postacrosomal sheath of bovine spermatozoa. Gamete Res 20:329-342. Oura C, Toshimori K (1990): Ultrastructural studies on the fertilization of mammalian ggmetes. Int Rev Cytol 122:105-151. Toshimori K, Araki S, Oura C (1988): Masking of sperm maturation
293
antigen by sialic acid in the epididymis of the mouse: An immunohistochemical study. Histochemistry 90: 195-200. Toshimori K, Araki S, Oura C (1990): Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface. Biol Reprod 42:151-160. Toshimori K, Higashi R, Oura C (1985): Distribution of intramembranous particles and filipin-sterol complexes in mouse sperm membranes: Polyene antibiotic filipin treatment. Am J Anat 174:455-470. Yanagimachi R (1988):Mammalian fertilization. In E Knobil, J Neill, et al. (eds): “The Physiology of Reproduction.” New York: Raven Press, pp 135-185.
