RELEASE OF HYALURONIDASE FROM GUINEA-PIG SPERMATOZOA THROUGH AN ACROSOME REACTION INITIATED BY CALCIUM B. JANE ROGERS and RYUZO YANAGIMACHI Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822, U.S.A.
(Received 10th December 1974) Hyaluronidase which is localized in the sperm acrosome (Mancini, Alonso, Barquet, Alvarez & Nemirovsky, 1964) is released by ageing of the spermatozoa (Mann, 1964), freeze-thawing (Ackerman, 1970) and treatment of spermatozoa with a variety of reagents such as digitonin (Austin, 1960), Hyamine 2389 (Hartree & Srivastava, 1965) and MgCl2 (Srivastava, 1973). These conditions seem to destroy the integrity of the plasma and acrosomal membranes (Srivastava, Munnell, Yang & Foley, 1974). The release of hyaluronidase from living spermatozoa is believed to occur through a process called the acrosome reaction. This reaction, which involves multiple fusions between the outer acrosomal membrane and overlying sperm plasma membrane (Barros, Bedford, Franklin & Austin, 1967), occurs only after the spermatozoa attain a `capacitated' state (Bedford, 1970). In fertilization, the functional significance of the acrosome reaction is probably that it allows the acrosomal enzymes to become accessible for digestion of the cumulus matrix and other egg envelopes as capacitated spermatozoa pass through these barriers (Austin, 1961 ; Bedford, 1970; Yanagimachi 1973). Attempts have been made by several workers to measure the hyaluronidase released from rabbit, hamster, and guinea-pig spermatozoa (Lewis & Ketchel, 1972; Rogers & Morton, 1973; Talbot & Franklin, 1974a, b), but they were confronted with difficulties in differentiating the hyaluronidase released from moribund or dead spermatozoa from that released as the result of an acrosome reaction in live spermatozoa. Yanagimachi & Usui (1974) developed a technique to induce a synchronous acrosome reaction of guinea-pig spermatozoa. According to them, guinea-pig spermatozoa incubated in Ca-free media undergo capacitation and are arrested in the capacitated state. If calcium ions are now added, the acrosome reaction is induced in a large proportion of spermatozoa within 10 min. The object of this study was to measure the release of hyaluronidase in such a sperm-capacitating system and attempt to correlate it with the synchronous acrosome reaction. The medium used was Ca-free Tyrode's solution supple¬ mented with 1 mg bovine serum albumin/ml (Fraction V, Reheis/Armour, Chicago, Illinois), K-penicillin G (100 U/ml) and streptomycin (50 µg/ml). This medium, called Ca-free TA, was prepared immediately before use and sterilized by passage through a millipore filter. Guinea-pig spermatozoa from the cauda epididymidis were suspended in Ca-free TA at a concentration 135
Jane Rogers and Ryuzo Yanagimachi of 0-3 to 1·0 107 spermatozoa/ml. An aliquot (1 ml) of the suspension was put in a glass vial (2-5 cm diam.) with an air-tight cap and incubated in air at 37°C. For each experiment, at least four replicate vials were prepared. Motility, the acrosome reaction of spermatozoa, and the hyaluronidase released from the spermatozoa into the medium were determined (1) shortly after the start of the incubation, (2) after incubation for 16£ to 24 hr, and (3) 10 min after the subsequent addition of calcium chloride. The analyses consisted of (a) examina¬ tion of the suspensions microscopically with dark-field illumination in order to estimate the % of spermatozoa that were motile and the % that had undergone the acrosome reaction, and (b) assays of the released hyaluronidase activity. For the latter, the remaining suspension was centrifuged at 600 g for 5 min and the hyaluronidase in the clear fluid was determined by the method of Rogers & Morton (1973). Hyaluronidase activities are expressed as a % of the amount liberated when the spermatozoa were treated with 1 % Triton X-100. This amount, 177+12 mU/108 spermatozoa, was regarded as the total hyaluronidase activity since Triton disrupted almost instantaneously all the acrosomes in the suspension. The hyaluronidase units were expressed in terms of nmol n-acetylglucosamine released/min. The level of hyaluronidase extractable with Triton X-100 was regarded as 100% of that which spermatozoa carry and was compared with the level of hyaluronidase released from spermatozoa during incubation. Release of hyaluronidase from spermatozoa was measured before and after an 136
.
incubation in Ca-free TA for 16^ to 24 hr. At the start of the incubation when 90 to 95% of the spermatozoa were motile, the extracellular hyaluronidase levels ranged from 22 to 26% (five experiments) of the total extractable with Triton. At the end of the incubations, when 70 to 80% of the spermatozoa were motile, the hyaluronidase levels had increased to between 30 and 39% (five experiments). This release of hyaluronidase before and during incubation is apparently not due to a live acrosome reaction, since such reactions do not occur in Ca-free TA, but appears to be due to acrosomal disruption accompanied by sperm death. To study if release of hyaluronidase was concomitant with the acrosome reaction, spermatozoa were first incubated in Ca-free TA for 15J to 22 hr and an aliquot (0-05 ml) of 0-1 M-calcium chloride solution was then added to give a final concentration of 5 mu. Extracellular hyaluronidase was measured before and about 10 min after the addition of calcium. The results (Table 1) showed that the mean level of hyaluronidase in the medium before addition of calcium was 32%; after calcium addition, the level increased to 64%. This correlates with a live acrosome reaction of 50% of the total sperm population. The surge of hyaluronidase release after the addition of calcium is obviously due to a synchronous acrosome reaction and not to post-mortem degeneration. To verify that calcium does not cause release of sperm hyaluronidase without the acrosome reaction and that calcium does not stimulate hyaluronidase activity, two controls were performed. For the first control, calcium (5 him) was added to sperm suspensions in Ca-free TA at 5 min and 3 hr after the start of incubation. At these times, no acrosome reaction was induced and no increase in extracellular hyaluronidase was found over the levels measured in the
137 Guinea-pig sperm hyaluronidase absence of calcium. For the second control, calcium (5 him) was added to supernatant samples from sperm suspensions which had previously been incubated for 5 min, 3 hr or 25 hr in Ca-free TA. No difference was found between hyaluronidase levels determined in the presence and absence of added calcium. It can be concluded that calcium does not stimulate the enzymatic activity of the hyaluronidase thus causing an apparent increase in extracellular hyaluronidase. Table 1. Release of
hyaluronidase by
No.
(xlO7)
Preincubation time (hr) in Ca-free medium
1 2 3
0-75 0-50 0-42 0-35 1-00 0-44
17 15-5 20 22 20 20
Sperm. Exp. concimi
4 5 6 *
The
an acrosome
reaction initiated
addition of calcium
At the end ofpreincubation in Ca-free medium
% Sperm,
% Sperm. with
acrosome
Extracellular
motile
reaction*
hyaluronidase^
80 75 80 70 80 75
0 0 0 0 0 0
29%
19% 38% 34%
39% 33%
At 10 min
% Sperm.
by
the
after addition of calcium % Sperm. with
acrosome
motile
reaction*
80 75 80 70 80 75
50 50 50 50 55 45
Extracellular
hyaluronidase 66% 50% 70% 61% 68% 69%
% of spermatozoa that had undergone an acrosome reaction was determined on the total
population. t Extracellular hyaluronidase X-100 in a duplicate sample. sperm
is
expressed
as
% of hyaluronidase
extractable with Triton
technique used in this study appears to be more reliable than those used previously (Rogers & Morton, 1973; Talbotíí al., 1974a, b) for the measurement of the release of hyaluronidase, since the potential hyaluronidase leakage due to sperm death is separated from the release of hyaluronidase mediated by the acrosome reaction. Although hyaluronidase is associated with the acrosome, its association with acrosomal membranes is controversial (Metz, Seiguer & Castro, 1972; McRorie & Williams, 1974). The measurement of hyaluronidase release as an index of the completion of capacitation might be applicable for The
human spermatozoa which have acrosome reactions that are difficult to observe. If the spermatozoa could tolerate a period of incubation in a calcium-free medium, the later addition of calcium chloride and estimation of released hyaluronidase might serve as a biochemical assay for capacitation. The % of motile spermatozoa would be carefully monitored before and after the addition of the calcium and an increase of hyaluronidase unaccompanied by cell death might be attributable to an acrosome reaction in capacitated sperma¬ tozoa. Even if some hyaluronidase is released into the medium before the addi¬ tion of calcium, a significant increase at the time of the addition could still be attributable to a live acrosome reaction and indicative of capacitation. A biochemical assay of capacitation of this sort would be useful in work on human reproduction for which eggs are not readily available.
bull, rabbit
or
138
. Jane
Rogers and Ryuzo Yanagimachi This study was supported by grants from NIH-USPHS (HD-03402), the Population Council and the Ford Foundation. One of us (B.J.R.) is a recipient of a NIH Child Health and Human Development post-doctoral fellowship (HD 55134-01). We wish to thank Cherrie A. Mahi for her assistance in pre¬ paring the manuscript. REFERENCES
Ackerman, D. R. (1970) Hyaluronidase in human semen and sperm suspensions subjected to tempera¬ ture shock and to freezing. J. Reprod. Fert. 23, 521-523. Austin, C. R. (1960) Capacitation and the release of hyaluronidase from spermatozoa. J. Reprod. Fert. 1, 310-311.
Austin, C. R. (1961) The Mammalian Egg. Blackwell Scientific Publications, Oxford. Barros, C, Bedford, J. M., Franklin, L. E. & Austin, C. R. (1967) Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell Biol. 34, C1-C5. Bedford, J. M. (1970) Sperm capacitation and fertilization in mammals. Biol. Reprod., Suppl. 2, 128-158.
& Srivastava, P. N. (1965) Chemical composition of the acrosomes of ram sperma¬ J. Reprod. Fert. 9, 47-60. Lewis, B. K. & Ketchel, M. M. (1972) Effects of female reproductive tract secretions on rabbit sperm. 1. Release of hyaluronidase in vitro. Proc. Soc exp. Biol. Med. 141, 712-718. Mancini, R. E., Alonso, ., Barq_uet, J., Alvarez, B. & Nemirovsky, M. (1964) Histo-immunological localization of hyaluronidase in the bull testis. J. Reprod. Fert. 8, 325-330. Mann, T. (1964) The Biochemistry of Semen and of the Male Reproductive Tract. Methuen, London. McRorie, R. A. & Williams, W. L. (1974) Biochemistry of mammalian fertilization. In Annual Review of Biochemistry, pp. 777—803. Ed. E. E. Snell. Annual Reviews, Inc., Palo Alto, California. Metz, C. B., Seiguer, A. C. & Castro, A. E. (1972) Inhibition of the cumulus dispersing and hya¬ luronidase activities of sperm by heterologous and isologous antisperm antibodies. Proc. Soc exp.
Hartree, E. R. tozoa.
Biol. Med. 140, 776-781. Rogers, B. J. & Morton, . E. (1973) The release of hyaluronidase from capacitating hamster sperma¬ tozoa. J. Reprod. Fert. 35, 477^87. Srivastava, P. . (1973) Removal of acrosomes of ram and rabbit spermatozoa. J. Reprod. Fert. 33,
323-326.
. & Foley, C W. (1974) Sequential release of acrosomal membranes and acrosomal enzymes of ram spermatozoa. J. Reprod. Fert. 35, 363-372. Talbot, P. & Franklin, L. E. (1974a) The release of hyaluronidase from guinea-pig spermatozoa during the course of the normal acrosome reaction in vitro. J. Reprod. Fert. 39, 429-432. Talbot, P. & Franklin, L. E. (1974b) Hamster sperm hyaluronidase. II. Its release from sperm in vitro in relation to the degenerative and normal acrosome reaction. J. exp. Z°°t· 189, 321-331. Yanagimachi, R. (1973) Behavior and functions of the structural elements of the mammalian sperm head in fertilization. In The Regulation of Mammalian Reproduction, pp. 215-230. Eds. S. J. Segal, R. Crozier, P. A. Corfman and P. G. Condliffe. Charles C. Thomas, Springfield, Illinois. Yanagimachi, R. & Usui, . (1974) Calcium dependence of the acrosome reaction and activation of guinea-pig spermatozoa. Expl Cell Res. 89, 161—174.
Srivastava, P. N., Munnell, J. F., Yang, C.
(Received 10th December 1974) Hyaluronidase which is localized in the sperm acrosome (Mancini, Alonso, Barquet, Alvarez & Nemirovsky, 1964) is released by ageing of the spermatozoa (Mann, 1964), freeze-thawing (Ackerman, 1970) and treatment of spermatozoa with a variety of reagents such as digitonin (Austin, 1960), Hyamine 2389 (Hartree & Srivastava, 1965) and MgCl2 (Srivastava, 1973). These conditions seem to destroy the integrity of the plasma and acrosomal membranes (Srivastava, Munnell, Yang & Foley, 1974). The release of hyaluronidase from living spermatozoa is believed to occur through a process called the acrosome reaction. This reaction, which involves multiple fusions between the outer acrosomal membrane and overlying sperm plasma membrane (Barros, Bedford, Franklin & Austin, 1967), occurs only after the spermatozoa attain a `capacitated' state (Bedford, 1970). In fertilization, the functional significance of the acrosome reaction is probably that it allows the acrosomal enzymes to become accessible for digestion of the cumulus matrix and other egg envelopes as capacitated spermatozoa pass through these barriers (Austin, 1961 ; Bedford, 1970; Yanagimachi 1973). Attempts have been made by several workers to measure the hyaluronidase released from rabbit, hamster, and guinea-pig spermatozoa (Lewis & Ketchel, 1972; Rogers & Morton, 1973; Talbot & Franklin, 1974a, b), but they were confronted with difficulties in differentiating the hyaluronidase released from moribund or dead spermatozoa from that released as the result of an acrosome reaction in live spermatozoa. Yanagimachi & Usui (1974) developed a technique to induce a synchronous acrosome reaction of guinea-pig spermatozoa. According to them, guinea-pig spermatozoa incubated in Ca-free media undergo capacitation and are arrested in the capacitated state. If calcium ions are now added, the acrosome reaction is induced in a large proportion of spermatozoa within 10 min. The object of this study was to measure the release of hyaluronidase in such a sperm-capacitating system and attempt to correlate it with the synchronous acrosome reaction. The medium used was Ca-free Tyrode's solution supple¬ mented with 1 mg bovine serum albumin/ml (Fraction V, Reheis/Armour, Chicago, Illinois), K-penicillin G (100 U/ml) and streptomycin (50 µg/ml). This medium, called Ca-free TA, was prepared immediately before use and sterilized by passage through a millipore filter. Guinea-pig spermatozoa from the cauda epididymidis were suspended in Ca-free TA at a concentration 135
Jane Rogers and Ryuzo Yanagimachi of 0-3 to 1·0 107 spermatozoa/ml. An aliquot (1 ml) of the suspension was put in a glass vial (2-5 cm diam.) with an air-tight cap and incubated in air at 37°C. For each experiment, at least four replicate vials were prepared. Motility, the acrosome reaction of spermatozoa, and the hyaluronidase released from the spermatozoa into the medium were determined (1) shortly after the start of the incubation, (2) after incubation for 16£ to 24 hr, and (3) 10 min after the subsequent addition of calcium chloride. The analyses consisted of (a) examina¬ tion of the suspensions microscopically with dark-field illumination in order to estimate the % of spermatozoa that were motile and the % that had undergone the acrosome reaction, and (b) assays of the released hyaluronidase activity. For the latter, the remaining suspension was centrifuged at 600 g for 5 min and the hyaluronidase in the clear fluid was determined by the method of Rogers & Morton (1973). Hyaluronidase activities are expressed as a % of the amount liberated when the spermatozoa were treated with 1 % Triton X-100. This amount, 177+12 mU/108 spermatozoa, was regarded as the total hyaluronidase activity since Triton disrupted almost instantaneously all the acrosomes in the suspension. The hyaluronidase units were expressed in terms of nmol n-acetylglucosamine released/min. The level of hyaluronidase extractable with Triton X-100 was regarded as 100% of that which spermatozoa carry and was compared with the level of hyaluronidase released from spermatozoa during incubation. Release of hyaluronidase from spermatozoa was measured before and after an 136
.
incubation in Ca-free TA for 16^ to 24 hr. At the start of the incubation when 90 to 95% of the spermatozoa were motile, the extracellular hyaluronidase levels ranged from 22 to 26% (five experiments) of the total extractable with Triton. At the end of the incubations, when 70 to 80% of the spermatozoa were motile, the hyaluronidase levels had increased to between 30 and 39% (five experiments). This release of hyaluronidase before and during incubation is apparently not due to a live acrosome reaction, since such reactions do not occur in Ca-free TA, but appears to be due to acrosomal disruption accompanied by sperm death. To study if release of hyaluronidase was concomitant with the acrosome reaction, spermatozoa were first incubated in Ca-free TA for 15J to 22 hr and an aliquot (0-05 ml) of 0-1 M-calcium chloride solution was then added to give a final concentration of 5 mu. Extracellular hyaluronidase was measured before and about 10 min after the addition of calcium. The results (Table 1) showed that the mean level of hyaluronidase in the medium before addition of calcium was 32%; after calcium addition, the level increased to 64%. This correlates with a live acrosome reaction of 50% of the total sperm population. The surge of hyaluronidase release after the addition of calcium is obviously due to a synchronous acrosome reaction and not to post-mortem degeneration. To verify that calcium does not cause release of sperm hyaluronidase without the acrosome reaction and that calcium does not stimulate hyaluronidase activity, two controls were performed. For the first control, calcium (5 him) was added to sperm suspensions in Ca-free TA at 5 min and 3 hr after the start of incubation. At these times, no acrosome reaction was induced and no increase in extracellular hyaluronidase was found over the levels measured in the
137 Guinea-pig sperm hyaluronidase absence of calcium. For the second control, calcium (5 him) was added to supernatant samples from sperm suspensions which had previously been incubated for 5 min, 3 hr or 25 hr in Ca-free TA. No difference was found between hyaluronidase levels determined in the presence and absence of added calcium. It can be concluded that calcium does not stimulate the enzymatic activity of the hyaluronidase thus causing an apparent increase in extracellular hyaluronidase. Table 1. Release of
hyaluronidase by
No.
(xlO7)
Preincubation time (hr) in Ca-free medium
1 2 3
0-75 0-50 0-42 0-35 1-00 0-44
17 15-5 20 22 20 20
Sperm. Exp. concimi
4 5 6 *
The
an acrosome
reaction initiated
addition of calcium
At the end ofpreincubation in Ca-free medium
% Sperm,
% Sperm. with
acrosome
Extracellular
motile
reaction*
hyaluronidase^
80 75 80 70 80 75
0 0 0 0 0 0
29%
19% 38% 34%
39% 33%
At 10 min
% Sperm.
by
the
after addition of calcium % Sperm. with
acrosome
motile
reaction*
80 75 80 70 80 75
50 50 50 50 55 45
Extracellular
hyaluronidase 66% 50% 70% 61% 68% 69%
% of spermatozoa that had undergone an acrosome reaction was determined on the total
population. t Extracellular hyaluronidase X-100 in a duplicate sample. sperm
is
expressed
as
% of hyaluronidase
extractable with Triton
technique used in this study appears to be more reliable than those used previously (Rogers & Morton, 1973; Talbotíí al., 1974a, b) for the measurement of the release of hyaluronidase, since the potential hyaluronidase leakage due to sperm death is separated from the release of hyaluronidase mediated by the acrosome reaction. Although hyaluronidase is associated with the acrosome, its association with acrosomal membranes is controversial (Metz, Seiguer & Castro, 1972; McRorie & Williams, 1974). The measurement of hyaluronidase release as an index of the completion of capacitation might be applicable for The
human spermatozoa which have acrosome reactions that are difficult to observe. If the spermatozoa could tolerate a period of incubation in a calcium-free medium, the later addition of calcium chloride and estimation of released hyaluronidase might serve as a biochemical assay for capacitation. The % of motile spermatozoa would be carefully monitored before and after the addition of the calcium and an increase of hyaluronidase unaccompanied by cell death might be attributable to an acrosome reaction in capacitated sperma¬ tozoa. Even if some hyaluronidase is released into the medium before the addi¬ tion of calcium, a significant increase at the time of the addition could still be attributable to a live acrosome reaction and indicative of capacitation. A biochemical assay of capacitation of this sort would be useful in work on human reproduction for which eggs are not readily available.
bull, rabbit
or
138
. Jane
Rogers and Ryuzo Yanagimachi This study was supported by grants from NIH-USPHS (HD-03402), the Population Council and the Ford Foundation. One of us (B.J.R.) is a recipient of a NIH Child Health and Human Development post-doctoral fellowship (HD 55134-01). We wish to thank Cherrie A. Mahi for her assistance in pre¬ paring the manuscript. REFERENCES
Ackerman, D. R. (1970) Hyaluronidase in human semen and sperm suspensions subjected to tempera¬ ture shock and to freezing. J. Reprod. Fert. 23, 521-523. Austin, C. R. (1960) Capacitation and the release of hyaluronidase from spermatozoa. J. Reprod. Fert. 1, 310-311.
Austin, C. R. (1961) The Mammalian Egg. Blackwell Scientific Publications, Oxford. Barros, C, Bedford, J. M., Franklin, L. E. & Austin, C. R. (1967) Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell Biol. 34, C1-C5. Bedford, J. M. (1970) Sperm capacitation and fertilization in mammals. Biol. Reprod., Suppl. 2, 128-158.
& Srivastava, P. N. (1965) Chemical composition of the acrosomes of ram sperma¬ J. Reprod. Fert. 9, 47-60. Lewis, B. K. & Ketchel, M. M. (1972) Effects of female reproductive tract secretions on rabbit sperm. 1. Release of hyaluronidase in vitro. Proc. Soc exp. Biol. Med. 141, 712-718. Mancini, R. E., Alonso, ., Barq_uet, J., Alvarez, B. & Nemirovsky, M. (1964) Histo-immunological localization of hyaluronidase in the bull testis. J. Reprod. Fert. 8, 325-330. Mann, T. (1964) The Biochemistry of Semen and of the Male Reproductive Tract. Methuen, London. McRorie, R. A. & Williams, W. L. (1974) Biochemistry of mammalian fertilization. In Annual Review of Biochemistry, pp. 777—803. Ed. E. E. Snell. Annual Reviews, Inc., Palo Alto, California. Metz, C. B., Seiguer, A. C. & Castro, A. E. (1972) Inhibition of the cumulus dispersing and hya¬ luronidase activities of sperm by heterologous and isologous antisperm antibodies. Proc. Soc exp.
Hartree, E. R. tozoa.
Biol. Med. 140, 776-781. Rogers, B. J. & Morton, . E. (1973) The release of hyaluronidase from capacitating hamster sperma¬ tozoa. J. Reprod. Fert. 35, 477^87. Srivastava, P. . (1973) Removal of acrosomes of ram and rabbit spermatozoa. J. Reprod. Fert. 33,
323-326.
. & Foley, C W. (1974) Sequential release of acrosomal membranes and acrosomal enzymes of ram spermatozoa. J. Reprod. Fert. 35, 363-372. Talbot, P. & Franklin, L. E. (1974a) The release of hyaluronidase from guinea-pig spermatozoa during the course of the normal acrosome reaction in vitro. J. Reprod. Fert. 39, 429-432. Talbot, P. & Franklin, L. E. (1974b) Hamster sperm hyaluronidase. II. Its release from sperm in vitro in relation to the degenerative and normal acrosome reaction. J. exp. Z°°t· 189, 321-331. Yanagimachi, R. (1973) Behavior and functions of the structural elements of the mammalian sperm head in fertilization. In The Regulation of Mammalian Reproduction, pp. 215-230. Eds. S. J. Segal, R. Crozier, P. A. Corfman and P. G. Condliffe. Charles C. Thomas, Springfield, Illinois. Yanagimachi, R. & Usui, . (1974) Calcium dependence of the acrosome reaction and activation of guinea-pig spermatozoa. Expl Cell Res. 89, 161—174.
Srivastava, P. N., Munnell, J. F., Yang, C.
