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Impaired antibody diffusion within the cytoplasmic membrane-fibrous sheath interspace of the human sperm tail
Journal of Reproductive Immunology, 23 (1993) 297-303 Elsevier Scientific Publishers Ireland Ltd.
291
JR1 00821
Brief Communication
Impaired antibody diffusion within the cytoplasmic membrane-fibrous sheath interspace of the human sperm tail Ali Jassima, Alan Grayb and Gian Franc0 Bottazzo” “Departments of Immunology and bMorbid Anatomy, London Hospital Medical College, Turner Street, London El 2AD (UK) (Accepted
for publication
3 March
1993)
Summary Monoclonal and polyclonal antibodies specific for the fibrous sheath (FS) were used to assess their diffusion within the subcytoplasmic membrane space of the human sperm tail. Using indirect immunofluorescence (IIF) and immunogold electron microscopy (IEM), the staining of the entire FS with these antibodies was observed only when the spermatozoa were completely demembranated by detergents or dried onto slides. Partial damage to sperm cytoplasmic membranes by freezing and thawing resulted in segmental staining of the FS. The IEM of such spermatozoa with the antibodies revealed the distribution of gold particles on the FS in denuded areas only and adjacent segments which retained their cytoplasmic membrane were not stained. These results indicate that partial disruption of the sperm plasma membranes does not lead to free diffusion of antibodies within the cytoplasmic membrane-FS interspace. This property appears to be unique to sperm cells and is due to their specialized structural organization. Key words: fibrous sheath; sperm cytoskeleton; sperm tail; immunogold electron microscopy: monoclonal antibodies Correspondence to: Ali Jassim, Street, London El 2AD, UK.
Department
0165-0378/93/$06.00 0 1993 Elsevier Printed and Published in Ireland
of Immunology,
Scientific
Publishers
London
Ireland
Hospital
Ltd.
Medical
College, Turner
298
In somatic cells, the cytoplasmic membrane is the only barrier between the external environment and cell interior and molecules which gain access through a defective plasma membrane diffuse freely within the cytoplasm. This is a well known phenomenon which has been utilized in the application of microinjection techniques for the demonstration, for example, of the effects of monoclonal antibodies (MoAbs) on the structural organization of the various components of the cytoskeletal network (Mangeat and Burridge, 1984). For spermatozoa, however, the microinjection technique is difficult to apply and, consequently, a different approach using antibodies was followed to probe the subcytoplasmic membrane space which separates between the plasma membrane and FS which possibly represents a modified form of intermediate filament (Jassim et al., 1992). The results described below show that disruption of the sperm cytoplasmic membrane at certain areas does not allow free diffusion of antibodies in the subcytoplasmic membrane space. This is due to the unique structural organization of the spermatozoa which evolves during spermiogenesis. Seminal samples were obtained from ten normozoospermic donors and the MoAbs RT97 and GDA-J/F3, which react with phosphorylated and nonphosphorylated FS products, respectively were selected for this study (Jassim et al., 1990, 1991; Jassim, 1991) together with FS-specific xenoantisera (Jassim et al., 1993). All the techniques used have previously been described (Jassim, 1991). During initial indirect immunofluorescence (IIF) investigations with GDA-J/F3, the disruption of sperm cytoplasmic membrane by repeated freezing and thawing in liquid nitrogen resulted in linear staining of the entire principal piece in the majority of sperm tails (Figs. l a,b). However, in about 5% of spermatozoa, the principal piece was only partially stained (Fig. 1), and the following possible mechanisms were considered: (a) lack of GDA-J/F3 antigen expression in the areas which were not stained, but this was unlikely as similar patterns of immunofluorescence were obtained with RT97 and with the mouse anti-FS xenoantisera (data not shown); (b) presence of localized anatomical defects in the FS structure; this was ruled out as treatment of the same sperm samples with Triton X-100 or drying them onto slides eliminated the partial staining (Fig. 2); and (c) impaired antibody diffusion within the subcytoplasmic membrane space. This last possibility was investigated by screening GDA-J/F3 with sperm whose cytoplasmic membranes were partially damaged by freezing the whole semen at -40°C (for 2 h or longer up to 2 months) and then thawing. Using IIF, the majority of such spermatozoa were observed to be stained at their principal piece, but the number of those with partial staining increased to 20-30'/0. Similar results were also obtained by direct immunofluorescence microscopy using fluorescein isothiocyanate (FITC)-conjugated GDA-J/F3. The IEM of the same samples showed a number of spermatozoa in which the distribution
299
v
\
i
J Fig. 1. Indirect immunofluorescence of anti-fibrous sheath GDA-J/F3 MoAb with human spermatozoa following the disruption of their cytoplasmic membranes by repeated freezing and thawing in liquid nitrogen. In the majority of spermatozoa the whole principal piece was stained (a,b}, but around 5% of spermatozoa showed partial staining of the anterior (c,d), middle (e,f) or posterior segments of the principal piece (g,h) or sometimes the immunofluorescence was discontinuous (i,j). Similar results were obtained with RT97 MoAb and anti-FS xenoantisera. (k,l) negative controls. (a,c,e,g,i,k) phase contrast, (b,d,f,h,j,l) fluorescence microscopy, x 400.
of gold particles was restricted to the denuded FS areas, and FS segments which retained their cell membranes were not stained (Fig. 3a,a'), thus indicating that although the cytoplasmic membrane was disrupted over several sizeable areas the antibody diffusion through the subcytoplasmic space was impeded. To confirm these results, the cytoplasmic membranes of the same sperm samples, i.e. those frozen at -40°C were solubilized with 1% Triton X100 and screened by IIF and IEM. Both tests revealed reactions with the entire principal piece and FS and no partial staining was observed (Fig. 3b). The importance of the structural organization of the sperm tail in impairing the antibody diffusion within the subcytoplasmic space was best il-
300
Fig. 2. Indirect immunofluorescence of GDA-J/F3 MoAb with 1% Triton X-100-treated spermatozoa (a,b) or those dried onto slides (c,d). Note that the antibody stained the entire principal piece of the tail and no partial staining was observed. (a,c) phase contrast, (b,d) immunofluorescence microscopy. (x 400).
lustrated in spermatids with aprotruded tails, i.e. those in which the tails enrolled within the cytoplasm instead of protruding as normal flagella (Jassim and Festenstein, 1988). A group of these cells were found in the same seminal samples which were frozen and thawed at -40°C. IEM screening of GDA-J/F3 with such spermatids whose cytoplasmic membranes were partially disrupted revealed exclusive distribution of gold particles on the FS surface which was not covered by the cytoplasmic membrane, while the FS surface facing the intact plasma membrane was not stained (Fig. 4). Thus, the cytoplasm of the round spermatids, like that of somatic cells, allowed free movement of proteins intracellularly and impairment of antibody diffusion occurred only in areas where the FS was in juxtaposition to the cytoplasmic membrane.
301
Fig. 3. Pre-embedding irmnunogold electron microscopy of GDA-J/F3 MoAb with human spermatozoa which were partially damaged by freezing and thawing at -40°C either alone (a,a’), or after further treatment with 1% Triton (b). Note the restricted distribution of the gold particles to the denuded FS repions, while those which retained their cytoplasmic membrane were not stained; both cross- and longitudinal sections are shown (a,a’). Following treatment of the same samples with 1% Triton X-100, which solubilized the cytoplasmic membranes, the reaction of the antibody was with the entire FS, and no partial staining was observed (b). fs: fibrous sheath; pm: plasma membrane. (a: x 90 000; a’: x 42 000; b: x 54 000).
The data presented here clearly show that in partially damaged sperm, the partial staining of their FS resulted from impaired antibody diffusion within the subcytoplasmic membrane space owing to the structural organization of the sperm tail. In the sperm tail, the 4-5 nm thick subcytoplasmic filaments (Escalier, 1984) which cross-link the plasma membrane to the FS could have contributed to the impairment of antibody diffusion through the plasma
302
Fig. 4. Pre-embedding immunogold electron microscopy of GDA-J/F3 MoAb with human germ cells which were partially damaged by freezing and thawing at -40°C. A binucleated round spermatid in which the tail failed to protrude as a normal flagellum, i,e. tail aprotrusion, is shown to reveal the reactivity of GDA-J/F3 MoAb with the FS. The FS was intensely stained on its internal surface while the surface in juxtaposition to the cytoplasmic membrane was unstained (a: x 27 000). (b) the same section at higher magnification ( x 60 000). fs: fibrous sheath; n: nucleus; pm: plasma membrane.
membrane-FS interspace. However, it is likely that the subcytoplasmic membrane space, which has an apparent width of about 10 nm (according to morphometric measurements of the electron micrograph), has impeded the movement of the 10-nm gold particles-conjugated antibodies. Furthermore, the antibodies used in both IIF and IEM must also have contributed to the ultimate size of the resultant complexes as the diameter of an IgG molecule has been estimated to be around 8 nm (Roth, 1982). However, the impaired diffusion of the FITC-GDA-J/F3 used in the direct immunofluorescence microscopy indicates that the subcytoplasmic space does not allow the free movement of single 8 nm IgG molecules, and further studies are needed to investigate whether smaller molecules are also impeded. Therefore, the mechanism of the impaired antibody diffusion seems to be at least partly physical, resulting from the close proximity of the cytoplasmic membrane to the FS.
303
This unique slim-fit structural organization of the sperm tail as characterized by the intimate FS-cytoplasmic membrane association occurs in development during the formation of the sperm tail as a result of deposition of the FS anlagen underneath the cytoplasmic membrane (Irons and Clermont, 1982). However, sperm development also involves the shedding of surplus cytoplasm which is cast off as residual bodies. This is important to rid the actively motile spermatozoa from any unnecessary extra weight. Also, the shedding of excess cytoplasm results in close approximation of the various flagellar structures to increase their stability during sperm motility. Furthermore, and as shown in this study, the close association of the FS and the cytoplasmic membrane impairs the free movement of molecules (immunoglobulins) within the subcytoplasmic membrane space. This could be of some biological significance, acting as a 'barrier' to prevent noxious substances from gaining access into the motor apparatus (axoneme) should there be a breach in the integrity of the cytoplasmic membrane of the actively beating flagella. Alternatively, this cytoplasmic compartmentalization could be useful for segregating certain bioactive molecules such as enzymes as the highly insoluble FS is less likely to allow large molecules to pass through. References Escalier, D. (1984) The cytoplasmic matrix of the human spermatozoon: Cross-filaments link the various cell components. Biol. Cell 51, 347-364. Irons, M.J. and Clermont, Y. (1982) Kinetics of fibrous sheath formation in the rat spermatid. Am. J. Anat. 165, 121-130. Jassim, A. (1991) A J-p97: A novel antigen of the human sperm tail fibrous sheath detected by a neurofilament monoclonal antibody. J. Reprod. Immunol. 20, 15-26. Jassim, A. and Festenstein, H. (1987) Molecular dissection of human testicular germ cell differentiation with monoclonal antibodies. J. Repro& Immunol. 12, 173-189. Jassim, A. and Festenstein, H. (1988) Identification of spermatids with aprotruded tails in human semen by monoclonal antibodies and electron microscopy. In: Molecular and Cellular Endocrinology of the Testis (Cooke, B.A. and Sharpe, R.M., eds.), pp. 203-208. Raven Press, New York. Jassim, A., Auger, D., Oliver, R.T.D. and Sachs, J.A. (1990) GDA-J/F3 monoclonal antibody as a novel probe for the human sperm tail fibrous sheath and its anomalies. Hum. Reprod. 5, 990-996. Jassim, A., Gillott, D. and AI-Zuhdi, Y. (1991) Human sperm tail fibrous sheath undergoes phosphorylation during its development. Hum. Reprod. 6, 1135-1142. Jassim, A., Gillott, D., AI-Zuhdi, Y., Gray, A., Foxon, R. and Bottazzo, G.F. (1992) Isolation and biochemical characterisation of the human sperm tail fibrous sheath. Hum. Reprod. 7, 86-94 Jassim, A., Gray, A. and AI-Zuhdi, Y. (1993) Antigenic determinants of human sperm tail fibrous sheath proteins. J. Reprod. Immunol. (in press). Mangeat, P.H. and Burridge, K. (1984) Immunoprecipitation of non-erythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures. J. Cell Biol. 98, 1363-1377. Roth, J, (1982) The protein A-Gold (pAG) technique: A qualitative and quantitative approach for antigen localization on thin sections. In: Techniques in immunocytochemistry, Vol. 1 (Bullock, G. and Pelruz, P., eds.), p. 108. Academic Press.
291
JR1 00821
Brief Communication
Impaired antibody diffusion within the cytoplasmic membrane-fibrous sheath interspace of the human sperm tail Ali Jassima, Alan Grayb and Gian Franc0 Bottazzo” “Departments of Immunology and bMorbid Anatomy, London Hospital Medical College, Turner Street, London El 2AD (UK) (Accepted
for publication
3 March
1993)
Summary Monoclonal and polyclonal antibodies specific for the fibrous sheath (FS) were used to assess their diffusion within the subcytoplasmic membrane space of the human sperm tail. Using indirect immunofluorescence (IIF) and immunogold electron microscopy (IEM), the staining of the entire FS with these antibodies was observed only when the spermatozoa were completely demembranated by detergents or dried onto slides. Partial damage to sperm cytoplasmic membranes by freezing and thawing resulted in segmental staining of the FS. The IEM of such spermatozoa with the antibodies revealed the distribution of gold particles on the FS in denuded areas only and adjacent segments which retained their cytoplasmic membrane were not stained. These results indicate that partial disruption of the sperm plasma membranes does not lead to free diffusion of antibodies within the cytoplasmic membrane-FS interspace. This property appears to be unique to sperm cells and is due to their specialized structural organization. Key words: fibrous sheath; sperm cytoskeleton; sperm tail; immunogold electron microscopy: monoclonal antibodies Correspondence to: Ali Jassim, Street, London El 2AD, UK.
Department
0165-0378/93/$06.00 0 1993 Elsevier Printed and Published in Ireland
of Immunology,
Scientific
Publishers
London
Ireland
Hospital
Ltd.
Medical
College, Turner
298
In somatic cells, the cytoplasmic membrane is the only barrier between the external environment and cell interior and molecules which gain access through a defective plasma membrane diffuse freely within the cytoplasm. This is a well known phenomenon which has been utilized in the application of microinjection techniques for the demonstration, for example, of the effects of monoclonal antibodies (MoAbs) on the structural organization of the various components of the cytoskeletal network (Mangeat and Burridge, 1984). For spermatozoa, however, the microinjection technique is difficult to apply and, consequently, a different approach using antibodies was followed to probe the subcytoplasmic membrane space which separates between the plasma membrane and FS which possibly represents a modified form of intermediate filament (Jassim et al., 1992). The results described below show that disruption of the sperm cytoplasmic membrane at certain areas does not allow free diffusion of antibodies in the subcytoplasmic membrane space. This is due to the unique structural organization of the spermatozoa which evolves during spermiogenesis. Seminal samples were obtained from ten normozoospermic donors and the MoAbs RT97 and GDA-J/F3, which react with phosphorylated and nonphosphorylated FS products, respectively were selected for this study (Jassim et al., 1990, 1991; Jassim, 1991) together with FS-specific xenoantisera (Jassim et al., 1993). All the techniques used have previously been described (Jassim, 1991). During initial indirect immunofluorescence (IIF) investigations with GDA-J/F3, the disruption of sperm cytoplasmic membrane by repeated freezing and thawing in liquid nitrogen resulted in linear staining of the entire principal piece in the majority of sperm tails (Figs. l a,b). However, in about 5% of spermatozoa, the principal piece was only partially stained (Fig. 1), and the following possible mechanisms were considered: (a) lack of GDA-J/F3 antigen expression in the areas which were not stained, but this was unlikely as similar patterns of immunofluorescence were obtained with RT97 and with the mouse anti-FS xenoantisera (data not shown); (b) presence of localized anatomical defects in the FS structure; this was ruled out as treatment of the same sperm samples with Triton X-100 or drying them onto slides eliminated the partial staining (Fig. 2); and (c) impaired antibody diffusion within the subcytoplasmic membrane space. This last possibility was investigated by screening GDA-J/F3 with sperm whose cytoplasmic membranes were partially damaged by freezing the whole semen at -40°C (for 2 h or longer up to 2 months) and then thawing. Using IIF, the majority of such spermatozoa were observed to be stained at their principal piece, but the number of those with partial staining increased to 20-30'/0. Similar results were also obtained by direct immunofluorescence microscopy using fluorescein isothiocyanate (FITC)-conjugated GDA-J/F3. The IEM of the same samples showed a number of spermatozoa in which the distribution
299
v
\
i
J Fig. 1. Indirect immunofluorescence of anti-fibrous sheath GDA-J/F3 MoAb with human spermatozoa following the disruption of their cytoplasmic membranes by repeated freezing and thawing in liquid nitrogen. In the majority of spermatozoa the whole principal piece was stained (a,b}, but around 5% of spermatozoa showed partial staining of the anterior (c,d), middle (e,f) or posterior segments of the principal piece (g,h) or sometimes the immunofluorescence was discontinuous (i,j). Similar results were obtained with RT97 MoAb and anti-FS xenoantisera. (k,l) negative controls. (a,c,e,g,i,k) phase contrast, (b,d,f,h,j,l) fluorescence microscopy, x 400.
of gold particles was restricted to the denuded FS areas, and FS segments which retained their cell membranes were not stained (Fig. 3a,a'), thus indicating that although the cytoplasmic membrane was disrupted over several sizeable areas the antibody diffusion through the subcytoplasmic space was impeded. To confirm these results, the cytoplasmic membranes of the same sperm samples, i.e. those frozen at -40°C were solubilized with 1% Triton X100 and screened by IIF and IEM. Both tests revealed reactions with the entire principal piece and FS and no partial staining was observed (Fig. 3b). The importance of the structural organization of the sperm tail in impairing the antibody diffusion within the subcytoplasmic space was best il-
300
Fig. 2. Indirect immunofluorescence of GDA-J/F3 MoAb with 1% Triton X-100-treated spermatozoa (a,b) or those dried onto slides (c,d). Note that the antibody stained the entire principal piece of the tail and no partial staining was observed. (a,c) phase contrast, (b,d) immunofluorescence microscopy. (x 400).
lustrated in spermatids with aprotruded tails, i.e. those in which the tails enrolled within the cytoplasm instead of protruding as normal flagella (Jassim and Festenstein, 1988). A group of these cells were found in the same seminal samples which were frozen and thawed at -40°C. IEM screening of GDA-J/F3 with such spermatids whose cytoplasmic membranes were partially disrupted revealed exclusive distribution of gold particles on the FS surface which was not covered by the cytoplasmic membrane, while the FS surface facing the intact plasma membrane was not stained (Fig. 4). Thus, the cytoplasm of the round spermatids, like that of somatic cells, allowed free movement of proteins intracellularly and impairment of antibody diffusion occurred only in areas where the FS was in juxtaposition to the cytoplasmic membrane.
301
Fig. 3. Pre-embedding irmnunogold electron microscopy of GDA-J/F3 MoAb with human spermatozoa which were partially damaged by freezing and thawing at -40°C either alone (a,a’), or after further treatment with 1% Triton (b). Note the restricted distribution of the gold particles to the denuded FS repions, while those which retained their cytoplasmic membrane were not stained; both cross- and longitudinal sections are shown (a,a’). Following treatment of the same samples with 1% Triton X-100, which solubilized the cytoplasmic membranes, the reaction of the antibody was with the entire FS, and no partial staining was observed (b). fs: fibrous sheath; pm: plasma membrane. (a: x 90 000; a’: x 42 000; b: x 54 000).
The data presented here clearly show that in partially damaged sperm, the partial staining of their FS resulted from impaired antibody diffusion within the subcytoplasmic membrane space owing to the structural organization of the sperm tail. In the sperm tail, the 4-5 nm thick subcytoplasmic filaments (Escalier, 1984) which cross-link the plasma membrane to the FS could have contributed to the impairment of antibody diffusion through the plasma
302
Fig. 4. Pre-embedding immunogold electron microscopy of GDA-J/F3 MoAb with human germ cells which were partially damaged by freezing and thawing at -40°C. A binucleated round spermatid in which the tail failed to protrude as a normal flagellum, i,e. tail aprotrusion, is shown to reveal the reactivity of GDA-J/F3 MoAb with the FS. The FS was intensely stained on its internal surface while the surface in juxtaposition to the cytoplasmic membrane was unstained (a: x 27 000). (b) the same section at higher magnification ( x 60 000). fs: fibrous sheath; n: nucleus; pm: plasma membrane.
membrane-FS interspace. However, it is likely that the subcytoplasmic membrane space, which has an apparent width of about 10 nm (according to morphometric measurements of the electron micrograph), has impeded the movement of the 10-nm gold particles-conjugated antibodies. Furthermore, the antibodies used in both IIF and IEM must also have contributed to the ultimate size of the resultant complexes as the diameter of an IgG molecule has been estimated to be around 8 nm (Roth, 1982). However, the impaired diffusion of the FITC-GDA-J/F3 used in the direct immunofluorescence microscopy indicates that the subcytoplasmic space does not allow the free movement of single 8 nm IgG molecules, and further studies are needed to investigate whether smaller molecules are also impeded. Therefore, the mechanism of the impaired antibody diffusion seems to be at least partly physical, resulting from the close proximity of the cytoplasmic membrane to the FS.
303
This unique slim-fit structural organization of the sperm tail as characterized by the intimate FS-cytoplasmic membrane association occurs in development during the formation of the sperm tail as a result of deposition of the FS anlagen underneath the cytoplasmic membrane (Irons and Clermont, 1982). However, sperm development also involves the shedding of surplus cytoplasm which is cast off as residual bodies. This is important to rid the actively motile spermatozoa from any unnecessary extra weight. Also, the shedding of excess cytoplasm results in close approximation of the various flagellar structures to increase their stability during sperm motility. Furthermore, and as shown in this study, the close association of the FS and the cytoplasmic membrane impairs the free movement of molecules (immunoglobulins) within the subcytoplasmic membrane space. This could be of some biological significance, acting as a 'barrier' to prevent noxious substances from gaining access into the motor apparatus (axoneme) should there be a breach in the integrity of the cytoplasmic membrane of the actively beating flagella. Alternatively, this cytoplasmic compartmentalization could be useful for segregating certain bioactive molecules such as enzymes as the highly insoluble FS is less likely to allow large molecules to pass through. References Escalier, D. (1984) The cytoplasmic matrix of the human spermatozoon: Cross-filaments link the various cell components. Biol. Cell 51, 347-364. Irons, M.J. and Clermont, Y. (1982) Kinetics of fibrous sheath formation in the rat spermatid. Am. J. Anat. 165, 121-130. Jassim, A. (1991) A J-p97: A novel antigen of the human sperm tail fibrous sheath detected by a neurofilament monoclonal antibody. J. Reprod. Immunol. 20, 15-26. Jassim, A. and Festenstein, H. (1987) Molecular dissection of human testicular germ cell differentiation with monoclonal antibodies. J. Repro& Immunol. 12, 173-189. Jassim, A. and Festenstein, H. (1988) Identification of spermatids with aprotruded tails in human semen by monoclonal antibodies and electron microscopy. In: Molecular and Cellular Endocrinology of the Testis (Cooke, B.A. and Sharpe, R.M., eds.), pp. 203-208. Raven Press, New York. Jassim, A., Auger, D., Oliver, R.T.D. and Sachs, J.A. (1990) GDA-J/F3 monoclonal antibody as a novel probe for the human sperm tail fibrous sheath and its anomalies. Hum. Reprod. 5, 990-996. Jassim, A., Gillott, D. and AI-Zuhdi, Y. (1991) Human sperm tail fibrous sheath undergoes phosphorylation during its development. Hum. Reprod. 6, 1135-1142. Jassim, A., Gillott, D., AI-Zuhdi, Y., Gray, A., Foxon, R. and Bottazzo, G.F. (1992) Isolation and biochemical characterisation of the human sperm tail fibrous sheath. Hum. Reprod. 7, 86-94 Jassim, A., Gray, A. and AI-Zuhdi, Y. (1993) Antigenic determinants of human sperm tail fibrous sheath proteins. J. Reprod. Immunol. (in press). Mangeat, P.H. and Burridge, K. (1984) Immunoprecipitation of non-erythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures. J. Cell Biol. 98, 1363-1377. Roth, J, (1982) The protein A-Gold (pAG) technique: A qualitative and quantitative approach for antigen localization on thin sections. In: Techniques in immunocytochemistry, Vol. 1 (Bullock, G. and Pelruz, P., eds.), p. 108. Academic Press.