RAPID COMMUNICATION
ZIPGRAM
PROTEOLYTIC ACTIVITY OF GUINEA P I G SPERMATOZOA AFTER INDUCTION OF THE ACROSOMAL REACTION IN VITRO P . GADDUM-ROSSE AND R . J . BLANDAU Department of Biological S t r u c t u r e , School of Medicine, University of Washington, S e a t t l e , Washington 98195
ABSTRACT After inducing the acrosomal reaction in guinea pig spermatozoa in v i t r o , the sperm were t e s t e d f o r p r o t e o l y t i c a c t i v i t y by applying them t o membranes of fixed g e l a t i n . One t o 5% of them showed s l i g h t evidence of p r o t e o l y t i c a c t i v i t y , while t h e r e s t were completely negative. Sperm t h a t had retained t h e i r acrosomes t h r o u g h o u t the incubation period displayed intense p r o t e o l y t i c a c t i v i t y . These r e s u l t s suggest t h a t proteinases may be l o s t from spermatozoa as a r e s u l t of the acrosomal reaction. I t i s generally believed t h a t proteinases a r e of major importance f o r sperm penetration through the zona pellucida of t h e egg ( f o r review, see Zaneveld e t a l . , ' 7 5 ) .
Much of the outer acrosomal membrane, the overlying
plasma membrane and the acrosomal contents a r e l o s t before t h e spermatozoon e n t e r s t h e zona pellucida; only the inner acrosomal membrane and t h e equatorial segment remain a s t h e sperm begins penetration of the zona.
Thus i t has
been postulated t h a t any enzymatic "zona l y s i n " must be bound t o the inner acrosomal membrane o r equatorial segment (Bedford, '68; Yanagimachi and Noda, ' 7 0 ) .
Removal of the outer membranes of the sperm head by a r t i f i c i a l
means has provided some evidence f o r t h e presence of acrosin, a major acrosomal p r o t e o l y t i c enzyme, on the inner acrosomal membrane and/or equatorial
segment ( S c h i l l and Wolff, ' 7 4 ; Morton, '75; Zahler and Cloak, '75; Brown and Hartree, ' 7 6 ) .
I n none of these experiments, however, has p r o t e o l y t i c a c t i v i t y
been demonstrated in sperm t h a t have undergone the physiological acrosmal
423
reaction which precedes zona penetration. Recent advances in the techniques of f e r t i l i z a t i o n in v i t r o have made i t possible t o c o l l e c t , in rodent species a t l e a s t , spermatozoa in which capacitation and the acrosomal reaction have been completed.
I f acrosin i s
indeed bound t o the inner acrosomal membrane, i t should be c l e a r l y demonstrable on such sperm.
I n t h i s study we t e s t e d proteolytic a c t i v i t y
of guinea pig sperm, a f t e r induction of t h e acrosomal reaction in v i t r o , by applying them t o fixed g e l a t i n membranes.
With t h i s technique, proteolytic
a c t i v i t y i n individual spermatozoa i s indicated by digestion of the g e l a t i n s u b s t r a t e (Gaddum and Blandau, '70; Gaddum-Rosse and Blandau, ' 7 2 ) .
We
chose t o use guinea pig sperm because of t h e i r large, conspicuous acrosomes, and because the sperm capacitation techniques f o r t h i s species have been worked out i n d e t a i l (Yanagimachi, '72; Barros e t a l . , '73; Barros, ' 7 4 ) . I t i s known t h a t mammalian acrosomes contain p r o t e o l y t i c enzymes other than a c r o s i n , b u t these have n o t been f u l l y characterized (Zaneveld e t a l . , '75).
Although i t i s possible t h a t the g e l a t i n membrane technique demonstrates
an enzyme (or enzymes) other t h a n acrosin, the experiments of Wendtet a l . ( ' 7 5 a , b ) suggest t h a t t h e enzyme demonstrated by t h i s technique i s indeed
acrosin. MATERIALSANDMETHODS The acrosome reaction was induced in guinea pig spermatozoa in v i t r o by a technique based on t h a t of Yanagimachi ( '72) and of Barros e t a
.
('73).
The medium used f o r incubation was B r i n s t e r ' s
Medium f o r Ovum Cu ture, BMOC-2 ( B r i n s t e r , ' 6 9 ) .
Spermatozoa were recovered
from the caudae ep didymides of f r e s h l y k i l l e d male guinea pigs and suspended i n BMOC-2 a t 37' C
Preparations were s e t up i n s t e r i l e Maximow depression
s l i d e s , each preparation consisting of about 0.3 ml of suspension containing sperm in a concentration of 20-30 x 106 per ml.
424
The preparations were
immediately covered with mineral o i l and incubated f o r u p t o 20 hours a t 37' C .
They were observed periodically under a dissecting microscope t o
assess overall m o t i l i t y and, a t i n t e r v a l s , small samples were removed and examined under the phase c o n t r a s t microscope t o determine t h e proportion of sperm t h a t had undergone t h e acrosomal reaction.
When a high proportion of
such sperm was seen, small drops of t h e suspension were removed and applied t o gel a t i n membranes , s p e c i a l l y prepared a s described previously (Gaddum-Rosse and Blandau, ' 7 2 ) .
Each drop was spread rapidly over t h e membrane and
immediately covered with a coverslip, and the preparation was then sealed and incubated a t 37' C .
They were observed periodically under phase c o n t r a s t
objectives t o determine t h e percentage of sperm demonstrating l y s i s of the gelatin substrate. RESULTS
Shortly a f t e r the onset of incubation, the spermatozoa tended
t o form l a r g e aggregates, a phenomenon a l s o noted by Yanagimachi ( ' 7 2 ) and Barros e t a l . ( ' 7 3 ) . The clumped sperm did not disperse spontaneously, and pipetting of the suspensions a t i n t e r v a l s causedonly temporary d i s p e r s a l . By 9-10 hours, however, s i g n i f i c a n t numbers of sperm began t o detach themselves and swim f r e e l y .
This process continued over the next 10 hours.
During t h i s period t h e aggregates tended t o s e t t l e in t h e center of the depression s l i d e , a n d l a r g e numbers of free-swimming sperm could be found in the more peripheral areas.
Samples of these c e l l s could be obtained
e a s i l y by means of a f i n e p i p e t t e . Occurrence of the Acrosome Reaction.
When samples of peripheral,
free-swimming sperm were examined a t i n t e r v a l s under t h e phase c o n t r a s t microscope, i t was found t h a t the acrosomal reaction had occurred in a very few sperm ( l e s s than 1 % ) a f t e r 5-6 hours.
By 11-12 hours, samples from
most preparations indicated a high r a t e of reaction:
425
t y p i c a l l y , such samples
contained approximately 80% "acrosome-reacted", highly moti l e sperm, 10% motile sperm with i n t a c t acrosomes, and 10% immotile sperm lacking acrosomes. There was l i t t l e change in these r a t i o s in samples taken u p t o 17-18 hours. Subsequently, t h e proportion of immotile sperm increased rapidly.
This
time course corresponds t o t h a t reported by Yanagimachi ( ' 7 2 ) . Proteolytic Activity of Spermatozoa.
Once a sample of sperm was applied
t o a g e l a t i n membrane, m o t i l i t y was l o s t and i t became impossible t o d i f f e r e n t i a t e between sperm t h a t had undergone the t r u e acrosomal reaction and those t h a t had l o s t t h e i r acrosomes due t o degenerative changes.
Therefore, when samples were obtained f o r the membrane s t u d i e s , duplicate samples were always checked under phase objectives t o determine the percentage
of immotile sperm lacking acrosomes.
Only when the percentage of such
sperm was very low (10% o r l e s s ) were the samples used f o r t h e proteolytic activity studies. I t was necessary t o ensure t h a t a c e r t a i n number of i n t a c t , unreacted sperm were present in the sample t o serve as controls.
Therefore, samples
were usually taken a t 10-11 hours, when about 30% of the sperm s t i l l had i n t a c t acrosomes.
These control sperm showed strong proteolytic a c t i v i t y
on the g e l a t i n membranes ( f i g s . 1 , 2).
Clear zones, representing lysed g e l a t i n ,
F I G U R E LEGENDS
1 and 2 Guinea pig spermatozoa a f t e r four hours on fixed g e l a t i n membranes, following a preincubation period of 10-11 hours in BMOC-2 ( s e e t e x t ) . 1
A spermatozoon t h a t has retained i t s acrosome (on the r i g h t ) showing
strong proteolytic a c t i v i t y . The c l e a r zone around the sperm head i s due t o l y s i s o f the g e l a t i n s u b s t r a t e . The spermatozoon on the l e f t has no acrosome and shows no proteolytic a c t i v i t y . (The s l i g h t "halo" e f f e c t i s due t o the use of phase c o n t r a s t o b j e c t i v e s ) . X 1087. 2 A spermatozoon with an i n t a c t acrosome shows strong proteolytic a c t i v i t y . Close t o i t (arrowed) i s a spermatozoon t h a t has no acrosome and shows very weak proteolytic a c t i v i t y . X 1087.
426
developed around the acrosomal region of t h e sperm head and enlarged u n t i l the e n t i r e head and part of t h e flagellum were included.
The proteolytic
reaction was identical t o t h a t of fresh epididymal sperm, described in d e t a i l previously (Gaddum-Rosse and Blandau, ' 7 2 ) .
In c o n t r a s t , sperm in
which the acrosome was absent r a r e l y showed any v i s i b l e p r o t e o l y t i c a c t i v i t y ( f i g . 1 , sperm on the l e f t ) .
The great majority of these had undergone
the acrosomal reaction and had been highly motile j u s t p r i o r t o being placed on the membranes.
In only I-5% of them was there any c l e a r evidence o f
proteolytic a c t i v i t y :
these displayed a small c l e a r zone of lysed g e l a t i n
around the a n t e r i o r half of the head.
The maximum reaction obtained i s
shown in f i g u r e 2 b u t in the majority of cases, proteolysis was even l e s s extensive. DISCUSSION The r e s u l t s reported here indicate t h a t the g r e a t majority of guinea pig spermatozoa t h a t have undergone t h e acrosomal reaction in v i t r o do not display p r o t e o l y t i c a c t i v i t y on g e l a t i n membranes.
In c o n t r a s t ,
those t h a t have not y e t undergone t h e acrosomal reaction (and a r e present on the same membranes, s c a t t e r e d randomly among the "reacted" sperm) show strong proteolytic a c t i v i t y .
These findings suggest t h a t proteinases a r e l o s t
from guinea pig spermatozoa as a r e s u l t o f t h e acrosomal reaction.
We
recognize t h a t an absence of proteolytic a c t i v i t y on g e l a t i n membranes does not necessarily imply a loss of a l l p r o t e o l y t i c a c t i v i t y .
I t i s q u i t e possible,
f o r example, t h a t enzymes t i g h t l y bound t o the inneracrosomal membrane or equatorial segment may not be demonstrable by this technique, or t h a t d i f f e r e n t environmental conditions would be required t o accomplish t h e i r r e l e a s e (such a s a l t e r e d pH o r ionic c o n s t i t u t i o n of the f l u i d ) .
Nonetheless, t h e s t r i k i n g
difference in p r o t e o l y t i c behavior between sperm t h a t have undergone the acrosomal reaction and those t h a t have not indicates t h a t a d e f i n i t e change
428
in proteolytic status has occurred. It would be important to apply more rigorous biochemical techniques to test for proteolytic enzymes remaining on spermatozoa after capacitation and the acrosomal reaction. The presumed role of acrosin in zona penetration cannot be fully appreciated until its presence on the heads of capacitated, acrosome-reacted spermatozoa can be unequivocally established. ACKNOWLEDGMENTS The authors thank Lynn Langley for skilled technical assistance and Roy Hayashi for assistance in photography. The work was supported by NIH Grant HD-03752. LITERATURE CITED Barros, C. 1974 Capacitation of mammalian spermatozoa. Basic Life Sci., 4B: 3-24. Barros, C., M. Berrios and E. Herrera 1973 Capacitation in vitro of guinea pig spermatozoa in a saline solution. J. Reprod. Fert., 34: 547-549. Bedford, J. M. 1968 Ultrastructural changes in the sperm head during fertilization in the rabbit. Am. J. Anat., 123: 329-358. Brinster, R. L. 1969 Mammalian embryo culture. In: The Mammalian Oviduct, E.S.E. Hafez and R. J. Blandau, eds. University of Chicago Press, Chicago, pp. 419-444,Table 2. Brown, C.R., and E. F. Hartree 1976 Effects of acrosin inhibitors on the soluble and membrane-bound forms of ram acrosin, and a reappraisal of the role of the enzyme in fertilization. Hoppe-Seyler's Zeit. Physiol. Chem., 357: 57-65. Gaddum, P., and R. J. Blandau 1970 Proteolytic reaction of mammalian spermatozoa on gelatin membranes. Science, 170: 749-751. Gaddum-Rosse, P., and R. J . Blandau 1972 Comparative studies on the proteolysis of fixed gelatin membranes by mammalian sperm acrosomes. Am. J. Anat., 134: 133-144. Morton, D. B. 1975 Acrosomal enzymes: immunochemical localization of acrosin and hyaluronidase in ram spermatozoa. J. Reprod. Fert., 45: 375-378. Schill, W . B., and H. H. Wolff 1971 Ultrastructure of human sperm acrosome and determination of acrosin activity under conditions of semen preservation. Int. J. Fert., 19: 217-223. Wendt, V., W. Leidl and H. Fritz 1975a The lysis effect of bull spermatozoa on gelatin substrate film. Methodical investigations. Hoppe-Seyler's Zeit. Physiol. Chem., 356: 315-323. Wendt, V., W . Leidl and H. Fritz 1975b The influence of various proteinase inhibitors on the gelatinolytic effect of ejaculated and uterine boar spermatozoa. Hoppe-Seyler's Zeit. Physiol. Chem. , 356: 1073-1078. Yanagimachi, R. 1972 Fertilization of guinea pig eggs in vitro. Anat. Rec., 174: 9-20. 429
Yanagimachi, R . , and Y . D. Noda 1970 U l t r a s t r u c t u r a l changes i n the hamster sperm head during f e r t i l i z a t i o n . J . U l t r a s t r . Res., 31: 465-485. Zahler, W. L . , and G. A. Doak 1975 I s o l a t i o n o f t h e o u t e r acrosomal membrane from bull sperm. Biochim. Biophys. Acta, 406: 479-488. Zaneveld, L.J.D., K. L . Polakoski and G . F . B . Schumacher 1975 The p r o t e o l y t i c enzyme systems of mammalian g e n i t a l t r a c t s e c r e t i o n s and spermatozoa. In: Proteases and Biological Control. E . Reich, D. B. Rifkin and E. Shaw, eds. Cold Spring Harbor Conferences on Cell P r o l i f e r a t i o n , Volume 2 , pp. 683-706.
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ZIPGRAM
PROTEOLYTIC ACTIVITY OF GUINEA P I G SPERMATOZOA AFTER INDUCTION OF THE ACROSOMAL REACTION IN VITRO P . GADDUM-ROSSE AND R . J . BLANDAU Department of Biological S t r u c t u r e , School of Medicine, University of Washington, S e a t t l e , Washington 98195
ABSTRACT After inducing the acrosomal reaction in guinea pig spermatozoa in v i t r o , the sperm were t e s t e d f o r p r o t e o l y t i c a c t i v i t y by applying them t o membranes of fixed g e l a t i n . One t o 5% of them showed s l i g h t evidence of p r o t e o l y t i c a c t i v i t y , while t h e r e s t were completely negative. Sperm t h a t had retained t h e i r acrosomes t h r o u g h o u t the incubation period displayed intense p r o t e o l y t i c a c t i v i t y . These r e s u l t s suggest t h a t proteinases may be l o s t from spermatozoa as a r e s u l t of the acrosomal reaction. I t i s generally believed t h a t proteinases a r e of major importance f o r sperm penetration through the zona pellucida of t h e egg ( f o r review, see Zaneveld e t a l . , ' 7 5 ) .
Much of the outer acrosomal membrane, the overlying
plasma membrane and the acrosomal contents a r e l o s t before t h e spermatozoon e n t e r s t h e zona pellucida; only the inner acrosomal membrane and t h e equatorial segment remain a s t h e sperm begins penetration of the zona.
Thus i t has
been postulated t h a t any enzymatic "zona l y s i n " must be bound t o the inner acrosomal membrane o r equatorial segment (Bedford, '68; Yanagimachi and Noda, ' 7 0 ) .
Removal of the outer membranes of the sperm head by a r t i f i c i a l
means has provided some evidence f o r t h e presence of acrosin, a major acrosomal p r o t e o l y t i c enzyme, on the inner acrosomal membrane and/or equatorial
segment ( S c h i l l and Wolff, ' 7 4 ; Morton, '75; Zahler and Cloak, '75; Brown and Hartree, ' 7 6 ) .
I n none of these experiments, however, has p r o t e o l y t i c a c t i v i t y
been demonstrated in sperm t h a t have undergone the physiological acrosmal
423
reaction which precedes zona penetration. Recent advances in the techniques of f e r t i l i z a t i o n in v i t r o have made i t possible t o c o l l e c t , in rodent species a t l e a s t , spermatozoa in which capacitation and the acrosomal reaction have been completed.
I f acrosin i s
indeed bound t o the inner acrosomal membrane, i t should be c l e a r l y demonstrable on such sperm.
I n t h i s study we t e s t e d proteolytic a c t i v i t y
of guinea pig sperm, a f t e r induction of t h e acrosomal reaction in v i t r o , by applying them t o fixed g e l a t i n membranes.
With t h i s technique, proteolytic
a c t i v i t y i n individual spermatozoa i s indicated by digestion of the g e l a t i n s u b s t r a t e (Gaddum and Blandau, '70; Gaddum-Rosse and Blandau, ' 7 2 ) .
We
chose t o use guinea pig sperm because of t h e i r large, conspicuous acrosomes, and because the sperm capacitation techniques f o r t h i s species have been worked out i n d e t a i l (Yanagimachi, '72; Barros e t a l . , '73; Barros, ' 7 4 ) . I t i s known t h a t mammalian acrosomes contain p r o t e o l y t i c enzymes other than a c r o s i n , b u t these have n o t been f u l l y characterized (Zaneveld e t a l . , '75).
Although i t i s possible t h a t the g e l a t i n membrane technique demonstrates
an enzyme (or enzymes) other t h a n acrosin, the experiments of Wendtet a l . ( ' 7 5 a , b ) suggest t h a t t h e enzyme demonstrated by t h i s technique i s indeed
acrosin. MATERIALSANDMETHODS The acrosome reaction was induced in guinea pig spermatozoa in v i t r o by a technique based on t h a t of Yanagimachi ( '72) and of Barros e t a
.
('73).
The medium used f o r incubation was B r i n s t e r ' s
Medium f o r Ovum Cu ture, BMOC-2 ( B r i n s t e r , ' 6 9 ) .
Spermatozoa were recovered
from the caudae ep didymides of f r e s h l y k i l l e d male guinea pigs and suspended i n BMOC-2 a t 37' C
Preparations were s e t up i n s t e r i l e Maximow depression
s l i d e s , each preparation consisting of about 0.3 ml of suspension containing sperm in a concentration of 20-30 x 106 per ml.
424
The preparations were
immediately covered with mineral o i l and incubated f o r u p t o 20 hours a t 37' C .
They were observed periodically under a dissecting microscope t o
assess overall m o t i l i t y and, a t i n t e r v a l s , small samples were removed and examined under the phase c o n t r a s t microscope t o determine t h e proportion of sperm t h a t had undergone t h e acrosomal reaction.
When a high proportion of
such sperm was seen, small drops of t h e suspension were removed and applied t o gel a t i n membranes , s p e c i a l l y prepared a s described previously (Gaddum-Rosse and Blandau, ' 7 2 ) .
Each drop was spread rapidly over t h e membrane and
immediately covered with a coverslip, and the preparation was then sealed and incubated a t 37' C .
They were observed periodically under phase c o n t r a s t
objectives t o determine t h e percentage of sperm demonstrating l y s i s of the gelatin substrate. RESULTS
Shortly a f t e r the onset of incubation, the spermatozoa tended
t o form l a r g e aggregates, a phenomenon a l s o noted by Yanagimachi ( ' 7 2 ) and Barros e t a l . ( ' 7 3 ) . The clumped sperm did not disperse spontaneously, and pipetting of the suspensions a t i n t e r v a l s causedonly temporary d i s p e r s a l . By 9-10 hours, however, s i g n i f i c a n t numbers of sperm began t o detach themselves and swim f r e e l y .
This process continued over the next 10 hours.
During t h i s period t h e aggregates tended t o s e t t l e in t h e center of the depression s l i d e , a n d l a r g e numbers of free-swimming sperm could be found in the more peripheral areas.
Samples of these c e l l s could be obtained
e a s i l y by means of a f i n e p i p e t t e . Occurrence of the Acrosome Reaction.
When samples of peripheral,
free-swimming sperm were examined a t i n t e r v a l s under t h e phase c o n t r a s t microscope, i t was found t h a t the acrosomal reaction had occurred in a very few sperm ( l e s s than 1 % ) a f t e r 5-6 hours.
By 11-12 hours, samples from
most preparations indicated a high r a t e of reaction:
425
t y p i c a l l y , such samples
contained approximately 80% "acrosome-reacted", highly moti l e sperm, 10% motile sperm with i n t a c t acrosomes, and 10% immotile sperm lacking acrosomes. There was l i t t l e change in these r a t i o s in samples taken u p t o 17-18 hours. Subsequently, t h e proportion of immotile sperm increased rapidly.
This
time course corresponds t o t h a t reported by Yanagimachi ( ' 7 2 ) . Proteolytic Activity of Spermatozoa.
Once a sample of sperm was applied
t o a g e l a t i n membrane, m o t i l i t y was l o s t and i t became impossible t o d i f f e r e n t i a t e between sperm t h a t had undergone the t r u e acrosomal reaction and those t h a t had l o s t t h e i r acrosomes due t o degenerative changes.
Therefore, when samples were obtained f o r the membrane s t u d i e s , duplicate samples were always checked under phase objectives t o determine the percentage
of immotile sperm lacking acrosomes.
Only when the percentage of such
sperm was very low (10% o r l e s s ) were the samples used f o r t h e proteolytic activity studies. I t was necessary t o ensure t h a t a c e r t a i n number of i n t a c t , unreacted sperm were present in the sample t o serve as controls.
Therefore, samples
were usually taken a t 10-11 hours, when about 30% of the sperm s t i l l had i n t a c t acrosomes.
These control sperm showed strong proteolytic a c t i v i t y
on the g e l a t i n membranes ( f i g s . 1 , 2).
Clear zones, representing lysed g e l a t i n ,
F I G U R E LEGENDS
1 and 2 Guinea pig spermatozoa a f t e r four hours on fixed g e l a t i n membranes, following a preincubation period of 10-11 hours in BMOC-2 ( s e e t e x t ) . 1
A spermatozoon t h a t has retained i t s acrosome (on the r i g h t ) showing
strong proteolytic a c t i v i t y . The c l e a r zone around the sperm head i s due t o l y s i s o f the g e l a t i n s u b s t r a t e . The spermatozoon on the l e f t has no acrosome and shows no proteolytic a c t i v i t y . (The s l i g h t "halo" e f f e c t i s due t o the use of phase c o n t r a s t o b j e c t i v e s ) . X 1087. 2 A spermatozoon with an i n t a c t acrosome shows strong proteolytic a c t i v i t y . Close t o i t (arrowed) i s a spermatozoon t h a t has no acrosome and shows very weak proteolytic a c t i v i t y . X 1087.
426
developed around the acrosomal region of t h e sperm head and enlarged u n t i l the e n t i r e head and part of t h e flagellum were included.
The proteolytic
reaction was identical t o t h a t of fresh epididymal sperm, described in d e t a i l previously (Gaddum-Rosse and Blandau, ' 7 2 ) .
In c o n t r a s t , sperm in
which the acrosome was absent r a r e l y showed any v i s i b l e p r o t e o l y t i c a c t i v i t y ( f i g . 1 , sperm on the l e f t ) .
The great majority of these had undergone
the acrosomal reaction and had been highly motile j u s t p r i o r t o being placed on the membranes.
In only I-5% of them was there any c l e a r evidence o f
proteolytic a c t i v i t y :
these displayed a small c l e a r zone of lysed g e l a t i n
around the a n t e r i o r half of the head.
The maximum reaction obtained i s
shown in f i g u r e 2 b u t in the majority of cases, proteolysis was even l e s s extensive. DISCUSSION The r e s u l t s reported here indicate t h a t the g r e a t majority of guinea pig spermatozoa t h a t have undergone t h e acrosomal reaction in v i t r o do not display p r o t e o l y t i c a c t i v i t y on g e l a t i n membranes.
In c o n t r a s t ,
those t h a t have not y e t undergone t h e acrosomal reaction (and a r e present on the same membranes, s c a t t e r e d randomly among the "reacted" sperm) show strong proteolytic a c t i v i t y .
These findings suggest t h a t proteinases a r e l o s t
from guinea pig spermatozoa as a r e s u l t o f t h e acrosomal reaction.
We
recognize t h a t an absence of proteolytic a c t i v i t y on g e l a t i n membranes does not necessarily imply a loss of a l l p r o t e o l y t i c a c t i v i t y .
I t i s q u i t e possible,
f o r example, t h a t enzymes t i g h t l y bound t o the inneracrosomal membrane or equatorial segment may not be demonstrable by this technique, or t h a t d i f f e r e n t environmental conditions would be required t o accomplish t h e i r r e l e a s e (such a s a l t e r e d pH o r ionic c o n s t i t u t i o n of the f l u i d ) .
Nonetheless, t h e s t r i k i n g
difference in p r o t e o l y t i c behavior between sperm t h a t have undergone the acrosomal reaction and those t h a t have not indicates t h a t a d e f i n i t e change
428
in proteolytic status has occurred. It would be important to apply more rigorous biochemical techniques to test for proteolytic enzymes remaining on spermatozoa after capacitation and the acrosomal reaction. The presumed role of acrosin in zona penetration cannot be fully appreciated until its presence on the heads of capacitated, acrosome-reacted spermatozoa can be unequivocally established. ACKNOWLEDGMENTS The authors thank Lynn Langley for skilled technical assistance and Roy Hayashi for assistance in photography. The work was supported by NIH Grant HD-03752. LITERATURE CITED Barros, C. 1974 Capacitation of mammalian spermatozoa. Basic Life Sci., 4B: 3-24. Barros, C., M. Berrios and E. Herrera 1973 Capacitation in vitro of guinea pig spermatozoa in a saline solution. J. Reprod. Fert., 34: 547-549. Bedford, J. M. 1968 Ultrastructural changes in the sperm head during fertilization in the rabbit. Am. J. Anat., 123: 329-358. Brinster, R. L. 1969 Mammalian embryo culture. In: The Mammalian Oviduct, E.S.E. Hafez and R. J. Blandau, eds. University of Chicago Press, Chicago, pp. 419-444,Table 2. Brown, C.R., and E. F. Hartree 1976 Effects of acrosin inhibitors on the soluble and membrane-bound forms of ram acrosin, and a reappraisal of the role of the enzyme in fertilization. Hoppe-Seyler's Zeit. Physiol. Chem., 357: 57-65. Gaddum, P., and R. J. Blandau 1970 Proteolytic reaction of mammalian spermatozoa on gelatin membranes. Science, 170: 749-751. Gaddum-Rosse, P., and R. J . Blandau 1972 Comparative studies on the proteolysis of fixed gelatin membranes by mammalian sperm acrosomes. Am. J. Anat., 134: 133-144. Morton, D. B. 1975 Acrosomal enzymes: immunochemical localization of acrosin and hyaluronidase in ram spermatozoa. J. Reprod. Fert., 45: 375-378. Schill, W . B., and H. H. Wolff 1971 Ultrastructure of human sperm acrosome and determination of acrosin activity under conditions of semen preservation. Int. J. Fert., 19: 217-223. Wendt, V., W. Leidl and H. Fritz 1975a The lysis effect of bull spermatozoa on gelatin substrate film. Methodical investigations. Hoppe-Seyler's Zeit. Physiol. Chem., 356: 315-323. Wendt, V., W . Leidl and H. Fritz 1975b The influence of various proteinase inhibitors on the gelatinolytic effect of ejaculated and uterine boar spermatozoa. Hoppe-Seyler's Zeit. Physiol. Chem. , 356: 1073-1078. Yanagimachi, R. 1972 Fertilization of guinea pig eggs in vitro. Anat. Rec., 174: 9-20. 429
Yanagimachi, R . , and Y . D. Noda 1970 U l t r a s t r u c t u r a l changes i n the hamster sperm head during f e r t i l i z a t i o n . J . U l t r a s t r . Res., 31: 465-485. Zahler, W. L . , and G. A. Doak 1975 I s o l a t i o n o f t h e o u t e r acrosomal membrane from bull sperm. Biochim. Biophys. Acta, 406: 479-488. Zaneveld, L.J.D., K. L . Polakoski and G . F . B . Schumacher 1975 The p r o t e o l y t i c enzyme systems of mammalian g e n i t a l t r a c t s e c r e t i o n s and spermatozoa. In: Proteases and Biological Control. E . Reich, D. B. Rifkin and E. Shaw, eds. Cold Spring Harbor Conferences on Cell P r o l i f e r a t i o n , Volume 2 , pp. 683-706.
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![Induction of the acrosome reaction in guinea-pig spermatozoa in vitro by the Ca ionophore A23187 [proceedings].](/assets/img/hold.png)
