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Non specific tail-tail agglutination of mammalian spermatozoa
Experimental
654
Cell Research 38, 654-659 (1965)
NON SPECIFIC TAIL-TAIL AGGLUTINATION MAMMALIAN SPERMATOZOA
OF
J. M. BEDFORD Department
of Physiology,
Royal
Veterinary
College, London, England
Received November 23. 1961
STUDIESwith
the electron microscope have shown that the mammalian sperm cell is covered by one continuous plasma membrane, yet regional differences occur in the distribution of net charge and of certain antigens over the surface of the spermatozoon. Such asymmetry in arrangement of the components of the cell surface seems to play a considerable part in determining the form or mode of aggregation of spermatozoa under the different conditions in which agglutination may occur. Numerous investigators [B, 10, 11, 17, 221 have observed that washed ejaculate or epididymal spermatozoa of several mammalian species tend to agglutinate by the head region in physiological solutions such as Ringer, Tyrode, 0.9 per cent NaCl (Fig. 1). A somewhat greater tendency for head-head agglutination is seen if such spermatozoa are suspended in either normal heated isologous, homologous or heterologous serum. In these aggregates the sperm tails remain quite free and show persistent vigorous activity. Although some degree of tail-tail agglutination occurs occasionally between rabbit spermatozoa suspended in high titres of normal human serum [17], tail-tail agglutination is never observed between mature rabbit spermatozoa in electrolyte solutions, or in normal homologous or other heterologous sera. Tail-tail agglutination does occur readily, however, when rabbit or other spermatozoa are suspended in specific heterologous antisera. In specific antisera prepared against xvhole spermatozoa, aggregates are formed in which head-tail, head-head and in particular tail-tail agglutination is apparent. It was shown by Henle et crl. [lo], that this type of aggregation in antisera results from the presence of antibodies against tail and head specific antigens, and that after absorption of all but the tail specific antibody, only tail-tail agglutination then occurs. The presence of tail isoagglutinins has been reported in the mouse [13, 181 and in man [16, 211, but it has not been possible to elicit production of tail isoagglutinins in the rabbit, either by injection of spermatozoa alone [17!, or together with Freund’s adjuvant [8, 201. Experimental
Cell Research 38
Tail-tail
agglutination
af mammalian
spermatozoa
653
Thus, at present, it is recognised that head-head agglutination of mammalian spermatozoa may often be non-specific, whereas the occurrence of tail-tail agglutination, particularly in homologous or isologous serum, is generally considered to indicate and to depend upon the presence of specific antibody against tail antigens. It therefore seemed of interest to report the consistent occurrence of non-specific tail-tail agglutination of motile rabbit spermatozoa in two different situations, namely, between mature spermatozoa in isotonic media of low electrolyte content, and also between immature spermatozoa from the caput epididymis, suspended in an isotonic electrolyte solution containing normal isologous or homologous serum.
MATERIALS
AND
METHODS
To avoid contamination with secretions of the accessory glands, mature spermatozoa were taken from the vas deferens and cauda epididymis of fertile male rabbits; spermatozoa were obtained also from caput regions i-3, as designated by Nicander [15], and from the proximal half of the corpus epididymis. After collection, the spermatozoa were suspended in the various media described below, in concentrations of 4-6 million spermatozoa/ml. These were then incubated at 37”C, in a chamber formed by a glass slide and a coverslip supported by Vaseline wax. Observations were made wit11 a phase contrast microscope during the initial two hours of incubation. The media used in this study were: Ringer solution, Ringer solution + 50-90 per cent heated normal rabbit serum, and, 0.32 M solutions of sucrose, glucose and fructose in glass distilled water. The rabbit serum was obtained either from virgin females, or from fertile males, and was heated for 15 min at 56% before use. OBSERVATIONS
Tail-tail agglutination of low ionic strength
of mature
spermatozoa
in isotonic
media
Mature rabbit spermatozoa in isotonic solutions of sucrose, glucose or fructose, at 37”C, show active movement that is often sustained for 2-3 hr. Under these conditions, aggregation of motile spermatozoa by the tail region similar to the tail-tail agglutination seen in specific antisera, soon occurs (cf. Fig. 3 of Henle et al. [lo]). In these aggregates the sperm heads generally remain quite free (Fig. 2), in contrast to the behaviour of spermatozoa in electrolyte solutions or in serum (Fig. 1). After some time in the sugar solutions, and as motility wanes, the aggregates tend to form larger groups or nets in which some heads become enmeshed, and which present a similar appearance to the strings of spermatozoa formed in specific antisera (cf. Experimental
Cell Research 38
656
J. M. Bedford
Figs. 1 and 2 of Henle et cxZ.[lo]). It is apparent that in isotonic media of lo\\ electrolyte concentration, the surface of the sperm tail becomes attractive not only to other tail surfaces, but also to particulate matter in the medium. Such material adheres to the surface of the tail, but never to that of the sperm head. With increase in electrolyte concentration of the medium, the properties of the sperm surface in media of low ionic strength are reversed. Thus, if increasing amounts of Ringer solution are added to a suspension of motile spermatozoa in an isotonic solution of sucrose or glucose, the inception of head-head agglutination occurs concomitantly with increasing concentration of electrolyte. Tail-tail agglutination of immature Ringer/serum solutions
(cuput) spermatozoa in
Spermatozoa from segments 1-3 of the rabbit epididymis, suspended in Ringer solution at 37”C, show relatively poor motility with a slight tendency for head-head, but with no tail-tail agglutination. The addition of 50-90 per cent normal serum induces some improvement in the motility of these caput spermatozoa; although the addition of serum brings about little increase in the degree of head-head agglutination, many motile caput spermatozoa subsequently develop a tendency to adhere by the tail region. Under these circumstances the spermatozoa adhere either by the tips of their tails or, as sometimes occurs, the tails become aligned along their length (Fig. 1); occasionally, as seen in Fig. 3, tail-tail and head-head agglutination ma! involve pairs of spermatozoa. Ultimately, as the motility decreases, these sperm also tend to become aggregated in randomly arranged groups. In the more distal regions of the cuput beyond the tlexure (regions l-3), and in the proximal half of the corpus epididymis, the spermatozoa shovv a progressive increase in their capacity for head-head agglutination when released into physiological solutions. The capacity for head-head agglutination reaches a maximum in the distal part of region 7, as designated by Nicander [13!. Fig. l.-Typical head-head agglutination of motile Ringer -t 50 y, heated serum. Phase contrast: x 440.
spermatozoa
suspcndctl
in
Fig. 2.-Tail-tail agglutination of vigorous motile mature rabbit spermatozoa, 0.32 M sucrose solution. Note the absence of head involvement. Phase contrast:
suspcndetl x 750.
in
Figs. 3 and il.--Immature spermatozoa from Ringer + 50 % heated homologous serum. Note also the head region of the same spermatozoa. the length of the tails. Phase contrast: Fig. 3 Experimental
Cell Research
28
mature
rabbit
the caput epididymis of the rabbit, suspended in in Fig. 3 the adhesion of the distal tail region and In Fig. 4 the sperms have become adherent along x 440, Fig. 4 x 750.
Tail-tail
agglutination
of mammalian
spermnto:ocr
Experimenlal
657
Cell Research 38
655
J. M. Bedford DISCUSSION
Tail-tail agglutination of mammalian spermatozoa is a phenomenon normally associated with the presence of specific antibodies in the surrounding medium. The present observations show, however, that in certain environments which allow active motility of rabbit spermatozoa, mutual adhesion of tail surfaces may be induced in the absence of the ligands of specific antibody. One may deduce from this finding that, in normal physiological media, in which non-specific head-head agglutination readily occurs, the complete absence of tail-tail agglutination cannot be attributed solely to the potentially disruptive force of vigorous tail movement. The absence of tailtail agglutination of mature spermatozoa in normal physiological media must therefore reflect real differences in surface characteristics of the sperm head and tail, respectively. This supposition receives support from investigations of the behaviour of bull [l-1] and of ram and rabbit spermatozoa [a] in a D.C. electric field, which have shown that in physiological media at physiological pH, the sperm tail possesses a significantly greater net negative charge than does the head. The forces which influence and promote the adhesion of like cells arc not yet fully understood. Present evidence suggests however, that such forces bear a similarity, in part at least, to those invoked by Verwey and Overbeek [19] for the maintenance of stability of lyophobic colloid suspensions, and in particular that there exists an inverse relationship between adhesiveness and cell surface potential [l, 7, 91. Since reduction in ionic strength of the surrounding medium results in a marked increase in electrophoretic mobility of ram [‘L] and rabbit spermatozoa (unpublished), it seems probable that the increased surface potential, acting as a repulsive force, may well be great enough to prevent mutual adhesion of sperm heads in isotonic media of low ionic strength. \Jrhile it is not possible at present to explain the coincidental development of attractive properties of the tail surface in such an environment (Fig. 2), it is ncrertheless relevant to note that with isotonic reduction in ionic strength of the surrounding medium, the increase in sperm surface potential is distinctly non-uniform in character, in that the orientation of the spermatozoa in the electric field changes concomitantly from a tail-anode to a head-anode configuration. The expression of a relatively greater increase in potential of the surface of the head compared with that of the tail, cannot be accounted for merely by increase in width of the electrical double layer [19], which would increase in a uniform way. It must also reflect the interaction of free radicals such as amino or imino groups at the sperm surface, with solute molecules Experimental
Cell Research 38
Tail-tail
agglutination
of mammalian
spermatozoa
659
in the medium, in a manner similar to that outlined by Lewin [la]. The fact that such interaction evidently occurs to a greater extent over the head region, further emphasises the different character of the sperm surface over the head and tail, respectively. Changes in several characteristics of spermatozoa have been noted to occur during epididymal passage. Included among these are changes in the response of the plasma membrane of the sperm head to osmium fixation [3, 41, and changes in the electrophoretic behaviour of whole spermatozoa is]. The common occurrence of tail-tail adhesion (Figs. 3 anti 4) betlveen caput spermatozoa in physiological media containing normal serum, contrasts with the behaviour of mature spermatozoa in the same medium (Fig. 1) and adds support to the evidence quoted above for the existence of significant changes in the plasma membrane of spermatozoa during maturation in the epididymis. It is important in this context to remember, however, that the fertilising ability of spermatozoa depends on the maturation of a number of difrerent systems within the cell: thus although many spermatozoa released from the corpus epididymis show the capacity for head-head agglutination, virtually none of these spermatozoa are capable of fertilising an ovum (unpublished). I wish to thank Mr Derek Symons for his help with the photographs in Figs. 1 and 2, which were taken using a Zeiss microflash device. I would also like to thank Professor E. C. Amoroso, F.R.S. for his helpful criticism of the manuscript. REFERENCES E. J., JAMES, A. M. and LOWICK, J. H. B., Nature 177, 576 (1956). A. D., Proc. Roy. Sot., B, 155, 292 (1961). J. M., Proc. Vth Congr. Anim. Reprod., Trento. Vol. III, 395 (1964). J. Anat. In press (1965).
1. AMBROSE, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 1X. 19.
R.4NGHAM, BEDFORD,
__ --
Sature 200, 1178 (1963). M. C., .J. Gen. Physiol. 30, 321 (194i). Drw, K., Biol. Bull. 93, 259 (1947). EDWARDS, R. G., J. Reprod. Fertil. 1, 385 (1960). FAUK-FREMIET, E. and NICHITA, G., Ann. Physiol. 3, 247 (1927). HESLE, W., HEXLE, G. and CHAMBERS, L. A., J. Exptl Med. 68, 335 (1938). I
WANG,
VERWEY, E. Elsevier, 20. WEIL, A. J. 21. WILSON, L., 22. Y.~MANE, J.,
.J. W. and OVERBEEK, J. TH. G., Theory of the Stability of Lyophobic Amsterdam, 1948. and FINKLER, A. IX., Proc. Sot. Expil Biol. Med. 102, 624 (1959).
Colloids.
Proc. Sot. Exptl Biol. Med. 85, 652 (1954). J. Coil. Agr., Hokkaido Imp. Univ. 9, 161 (1921). Experimenfal
Cell Research 38
654
Cell Research 38, 654-659 (1965)
NON SPECIFIC TAIL-TAIL AGGLUTINATION MAMMALIAN SPERMATOZOA
OF
J. M. BEDFORD Department
of Physiology,
Royal
Veterinary
College, London, England
Received November 23. 1961
STUDIESwith
the electron microscope have shown that the mammalian sperm cell is covered by one continuous plasma membrane, yet regional differences occur in the distribution of net charge and of certain antigens over the surface of the spermatozoon. Such asymmetry in arrangement of the components of the cell surface seems to play a considerable part in determining the form or mode of aggregation of spermatozoa under the different conditions in which agglutination may occur. Numerous investigators [B, 10, 11, 17, 221 have observed that washed ejaculate or epididymal spermatozoa of several mammalian species tend to agglutinate by the head region in physiological solutions such as Ringer, Tyrode, 0.9 per cent NaCl (Fig. 1). A somewhat greater tendency for head-head agglutination is seen if such spermatozoa are suspended in either normal heated isologous, homologous or heterologous serum. In these aggregates the sperm tails remain quite free and show persistent vigorous activity. Although some degree of tail-tail agglutination occurs occasionally between rabbit spermatozoa suspended in high titres of normal human serum [17], tail-tail agglutination is never observed between mature rabbit spermatozoa in electrolyte solutions, or in normal homologous or other heterologous sera. Tail-tail agglutination does occur readily, however, when rabbit or other spermatozoa are suspended in specific heterologous antisera. In specific antisera prepared against xvhole spermatozoa, aggregates are formed in which head-tail, head-head and in particular tail-tail agglutination is apparent. It was shown by Henle et crl. [lo], that this type of aggregation in antisera results from the presence of antibodies against tail and head specific antigens, and that after absorption of all but the tail specific antibody, only tail-tail agglutination then occurs. The presence of tail isoagglutinins has been reported in the mouse [13, 181 and in man [16, 211, but it has not been possible to elicit production of tail isoagglutinins in the rabbit, either by injection of spermatozoa alone [17!, or together with Freund’s adjuvant [8, 201. Experimental
Cell Research 38
Tail-tail
agglutination
af mammalian
spermatozoa
653
Thus, at present, it is recognised that head-head agglutination of mammalian spermatozoa may often be non-specific, whereas the occurrence of tail-tail agglutination, particularly in homologous or isologous serum, is generally considered to indicate and to depend upon the presence of specific antibody against tail antigens. It therefore seemed of interest to report the consistent occurrence of non-specific tail-tail agglutination of motile rabbit spermatozoa in two different situations, namely, between mature spermatozoa in isotonic media of low electrolyte content, and also between immature spermatozoa from the caput epididymis, suspended in an isotonic electrolyte solution containing normal isologous or homologous serum.
MATERIALS
AND
METHODS
To avoid contamination with secretions of the accessory glands, mature spermatozoa were taken from the vas deferens and cauda epididymis of fertile male rabbits; spermatozoa were obtained also from caput regions i-3, as designated by Nicander [15], and from the proximal half of the corpus epididymis. After collection, the spermatozoa were suspended in the various media described below, in concentrations of 4-6 million spermatozoa/ml. These were then incubated at 37”C, in a chamber formed by a glass slide and a coverslip supported by Vaseline wax. Observations were made wit11 a phase contrast microscope during the initial two hours of incubation. The media used in this study were: Ringer solution, Ringer solution + 50-90 per cent heated normal rabbit serum, and, 0.32 M solutions of sucrose, glucose and fructose in glass distilled water. The rabbit serum was obtained either from virgin females, or from fertile males, and was heated for 15 min at 56% before use. OBSERVATIONS
Tail-tail agglutination of low ionic strength
of mature
spermatozoa
in isotonic
media
Mature rabbit spermatozoa in isotonic solutions of sucrose, glucose or fructose, at 37”C, show active movement that is often sustained for 2-3 hr. Under these conditions, aggregation of motile spermatozoa by the tail region similar to the tail-tail agglutination seen in specific antisera, soon occurs (cf. Fig. 3 of Henle et al. [lo]). In these aggregates the sperm heads generally remain quite free (Fig. 2), in contrast to the behaviour of spermatozoa in electrolyte solutions or in serum (Fig. 1). After some time in the sugar solutions, and as motility wanes, the aggregates tend to form larger groups or nets in which some heads become enmeshed, and which present a similar appearance to the strings of spermatozoa formed in specific antisera (cf. Experimental
Cell Research 38
656
J. M. Bedford
Figs. 1 and 2 of Henle et cxZ.[lo]). It is apparent that in isotonic media of lo\\ electrolyte concentration, the surface of the sperm tail becomes attractive not only to other tail surfaces, but also to particulate matter in the medium. Such material adheres to the surface of the tail, but never to that of the sperm head. With increase in electrolyte concentration of the medium, the properties of the sperm surface in media of low ionic strength are reversed. Thus, if increasing amounts of Ringer solution are added to a suspension of motile spermatozoa in an isotonic solution of sucrose or glucose, the inception of head-head agglutination occurs concomitantly with increasing concentration of electrolyte. Tail-tail agglutination of immature Ringer/serum solutions
(cuput) spermatozoa in
Spermatozoa from segments 1-3 of the rabbit epididymis, suspended in Ringer solution at 37”C, show relatively poor motility with a slight tendency for head-head, but with no tail-tail agglutination. The addition of 50-90 per cent normal serum induces some improvement in the motility of these caput spermatozoa; although the addition of serum brings about little increase in the degree of head-head agglutination, many motile caput spermatozoa subsequently develop a tendency to adhere by the tail region. Under these circumstances the spermatozoa adhere either by the tips of their tails or, as sometimes occurs, the tails become aligned along their length (Fig. 1); occasionally, as seen in Fig. 3, tail-tail and head-head agglutination ma! involve pairs of spermatozoa. Ultimately, as the motility decreases, these sperm also tend to become aggregated in randomly arranged groups. In the more distal regions of the cuput beyond the tlexure (regions l-3), and in the proximal half of the corpus epididymis, the spermatozoa shovv a progressive increase in their capacity for head-head agglutination when released into physiological solutions. The capacity for head-head agglutination reaches a maximum in the distal part of region 7, as designated by Nicander [13!. Fig. l.-Typical head-head agglutination of motile Ringer -t 50 y, heated serum. Phase contrast: x 440.
spermatozoa
suspcndctl
in
Fig. 2.-Tail-tail agglutination of vigorous motile mature rabbit spermatozoa, 0.32 M sucrose solution. Note the absence of head involvement. Phase contrast:
suspcndetl x 750.
in
Figs. 3 and il.--Immature spermatozoa from Ringer + 50 % heated homologous serum. Note also the head region of the same spermatozoa. the length of the tails. Phase contrast: Fig. 3 Experimental
Cell Research
28
mature
rabbit
the caput epididymis of the rabbit, suspended in in Fig. 3 the adhesion of the distal tail region and In Fig. 4 the sperms have become adherent along x 440, Fig. 4 x 750.
Tail-tail
agglutination
of mammalian
spermnto:ocr
Experimenlal
657
Cell Research 38
655
J. M. Bedford DISCUSSION
Tail-tail agglutination of mammalian spermatozoa is a phenomenon normally associated with the presence of specific antibodies in the surrounding medium. The present observations show, however, that in certain environments which allow active motility of rabbit spermatozoa, mutual adhesion of tail surfaces may be induced in the absence of the ligands of specific antibody. One may deduce from this finding that, in normal physiological media, in which non-specific head-head agglutination readily occurs, the complete absence of tail-tail agglutination cannot be attributed solely to the potentially disruptive force of vigorous tail movement. The absence of tailtail agglutination of mature spermatozoa in normal physiological media must therefore reflect real differences in surface characteristics of the sperm head and tail, respectively. This supposition receives support from investigations of the behaviour of bull [l-1] and of ram and rabbit spermatozoa [a] in a D.C. electric field, which have shown that in physiological media at physiological pH, the sperm tail possesses a significantly greater net negative charge than does the head. The forces which influence and promote the adhesion of like cells arc not yet fully understood. Present evidence suggests however, that such forces bear a similarity, in part at least, to those invoked by Verwey and Overbeek [19] for the maintenance of stability of lyophobic colloid suspensions, and in particular that there exists an inverse relationship between adhesiveness and cell surface potential [l, 7, 91. Since reduction in ionic strength of the surrounding medium results in a marked increase in electrophoretic mobility of ram [‘L] and rabbit spermatozoa (unpublished), it seems probable that the increased surface potential, acting as a repulsive force, may well be great enough to prevent mutual adhesion of sperm heads in isotonic media of low ionic strength. \Jrhile it is not possible at present to explain the coincidental development of attractive properties of the tail surface in such an environment (Fig. 2), it is ncrertheless relevant to note that with isotonic reduction in ionic strength of the surrounding medium, the increase in sperm surface potential is distinctly non-uniform in character, in that the orientation of the spermatozoa in the electric field changes concomitantly from a tail-anode to a head-anode configuration. The expression of a relatively greater increase in potential of the surface of the head compared with that of the tail, cannot be accounted for merely by increase in width of the electrical double layer [19], which would increase in a uniform way. It must also reflect the interaction of free radicals such as amino or imino groups at the sperm surface, with solute molecules Experimental
Cell Research 38
Tail-tail
agglutination
of mammalian
spermatozoa
659
in the medium, in a manner similar to that outlined by Lewin [la]. The fact that such interaction evidently occurs to a greater extent over the head region, further emphasises the different character of the sperm surface over the head and tail, respectively. Changes in several characteristics of spermatozoa have been noted to occur during epididymal passage. Included among these are changes in the response of the plasma membrane of the sperm head to osmium fixation [3, 41, and changes in the electrophoretic behaviour of whole spermatozoa is]. The common occurrence of tail-tail adhesion (Figs. 3 anti 4) betlveen caput spermatozoa in physiological media containing normal serum, contrasts with the behaviour of mature spermatozoa in the same medium (Fig. 1) and adds support to the evidence quoted above for the existence of significant changes in the plasma membrane of spermatozoa during maturation in the epididymis. It is important in this context to remember, however, that the fertilising ability of spermatozoa depends on the maturation of a number of difrerent systems within the cell: thus although many spermatozoa released from the corpus epididymis show the capacity for head-head agglutination, virtually none of these spermatozoa are capable of fertilising an ovum (unpublished). I wish to thank Mr Derek Symons for his help with the photographs in Figs. 1 and 2, which were taken using a Zeiss microflash device. I would also like to thank Professor E. C. Amoroso, F.R.S. for his helpful criticism of the manuscript. REFERENCES E. J., JAMES, A. M. and LOWICK, J. H. B., Nature 177, 576 (1956). A. D., Proc. Roy. Sot., B, 155, 292 (1961). J. M., Proc. Vth Congr. Anim. Reprod., Trento. Vol. III, 395 (1964). J. Anat. In press (1965).
1. AMBROSE, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 1X. 19.
R.4NGHAM, BEDFORD,
__ --
Sature 200, 1178 (1963). M. C., .J. Gen. Physiol. 30, 321 (194i). Drw, K., Biol. Bull. 93, 259 (1947). EDWARDS, R. G., J. Reprod. Fertil. 1, 385 (1960). FAUK-FREMIET, E. and NICHITA, G., Ann. Physiol. 3, 247 (1927). HESLE, W., HEXLE, G. and CHAMBERS, L. A., J. Exptl Med. 68, 335 (1938). I
WANG,
VERWEY, E. Elsevier, 20. WEIL, A. J. 21. WILSON, L., 22. Y.~MANE, J.,
.J. W. and OVERBEEK, J. TH. G., Theory of the Stability of Lyophobic Amsterdam, 1948. and FINKLER, A. IX., Proc. Sot. Expil Biol. Med. 102, 624 (1959).
Colloids.
Proc. Sot. Exptl Biol. Med. 85, 652 (1954). J. Coil. Agr., Hokkaido Imp. Univ. 9, 161 (1921). Experimenfal
Cell Research 38