ZIPGRAM
RAPID COMMUNICATION
ACROSIN DOES NOT APPEAR TO BE BOUND TO THE INNER ACROSOMAL MEMBRANE OF BULL SPERMATOZOA G A B R I E L E SHAMS-BORHAN, DANIEL HUNEAU A N D JACQUES-EDMOND F L ~ C H O N S t a t i o n c e n t r a l e de Physiologie animale, I.N.R.A. 78350 Jouy-en-Josas, France
ABSTRACT Ferritin-conjugated soybean trypsin i n h i b i t o r was used f o r t h e ul t r a s t r u c t u r a l l o c a l i z a t i o n of acrosin in bull spermatozoa following acrosomal disruption. The f e r r i t i n label was observed i n t h e a n t e r i o r segment of the acrosome in disrupted c e l l s only. Emptied acrosomes were labeled, mostly on the external surface of t h e i r outer membrane. Labeling was a l s o found on the material bound t o detached acrosomal caps. However, a t no time could the f e r r i t i n label be found on t h e inner acrosomal membrane. I t i s concluded t h a t acrosin a c t i v i t y i s n o t present on the inner acrosomal membrane b u t i s l o s t from t h e acrosomal matrix as t h e acrosomal reaction proceeds.
Acrosin ( E C 3-4-21-10), a sperm acrosomal proteinase, has been considered an e s s e n t i a l enzyme for the penetration of the zona pellucida by the spermatozoon during f e r t i l i z a t i o n (Meizel and Mukerji , '76; Yang e t a '76, Hartree, ' 7 7 ) .
I t was suggested by Bedford ( ' 7 2 ) t h a t some zona l y s i n
might remain on t h e inner acrosomal membrane a f t e r the acrosome r e a c t on. Subsequently, biochemical s t u d i e s showed t h a t acrosin could be recovered bound t o t h e sperm head in preparations of disrupted c e l l s (Brown and Hartree, '74; Schill and Wolff, ' 7 4 ) . However, t h e immunocytochemical labeling of acrosin, which i s r e s t r i c t e d t o the a n t e r i o r segment of t h e acrosome i n i n t a c t sperm, was progressively abolished in t h i s location during acrosomal disruption and l o s s o f t h e outer acrosomal membrane (F1 ;chon e t a1 . , ' 7 7 ) .
Moreover Gaddum-Rosse and B1 andau
( ' 7 7 ) reported t h a t spermatozoa which had undergone an acrosome reaction no
143
longer showed g e l a t i n o l y t i c a c t i v i t y , and Brown and Harrison ( '78) demonstrated t h a t acrosin i s readily released from sperm heads during proacrosin activation in a s a l i n e medium.
I n a f u r t h e r attempt t o check i f acrosin can be found on the inner acrosoma1 membrane, we have used a f e r r i t i n - l a b e l e d acrosin i n h i b i t o r on bull
spermatozoa a f t e r acrosomal disruption. MATERIAL AND METHODS
Ejaculated semen from b u l l s of proven f e r t i l i t y
was diluted w i t h 2 volumes of 0.15 M NaC1, 5 mM Hepes a t pH 7.5 and c e n t r i fuged through 1 . 3 M sucrose, 0.15 M NaCl a t 1,400 g f o r 25 minutes a t room temperature.
The sperm p e l l e t , a b o u t 0.2 ml, was resuspended in 3 ml o f
0.15 M NaC1, 0.005 M Hepes a t pH 7.5 t o y i e l d a f i n a l sperm concentration of about 2 x lo8 cells/ml
.
The acrosomes , which had been submitted t o t h i s
osmotic shock were f u r t h e r disrupted by passing the sperm c e l l s repeatedly through a Pasteur p i p e t t e (about 50 times).
The whole procedure was derived
from Zahler and Doak ( ' 7 5 ) .
After disruption, human serum albumin (2-20 mg/ml) was added t o prevent non-specific protein adsorption.
A1 iquots ( 0 . 5 ml) of the suspension were
incubated with 0.25 m l of 0 . 1 M T r i s H C l (pH 7.5) containing soybean trypsin i n h i b i t o r (Merck) t h a t had been conjugated with f e r r i t i n (Serva) using glutaraldehyde, according t o Kishida e t a1 . ( ' 7 5 ) .
The solution of conjugated
i n h i b i t o r (ferritin-SBTI) had an a c t i v i t y towards trypsin of 42 mUhl (using
benzoyl-L-arginine-p-nitroanilide as s u b s t r a t e ) and contained between 1 . 2 and 1.7 mg f e r r i t i n / m l .
Control a l i q u o t s were t r e a t e d with 0.15 M formate
buffer pH 2 . 4 a f t e r incubation in f e r r i t i n SBTI t o dissolve t h e acrosin (Shams-Borhan e t a l . , ' 7 6 ) .
Other a l i q u o t s were incubated in equivalent
amounts of unconjugated f e r r i t i n .
All samples were then washed twice with
0.15 m NaC1, 0.005 M Hepes a t pH 7.5, fixed with 1%glutaraldehyde a n d 1% 144
osmium t e t r o x i d e in 0.175 M cacodylate buffer and embedded in epon.
Thin
sections were observed e i t h e r unstained o r a f t e r uranyle a c e t a t e and lead c i t r a t e staining. RESULTS AND DISCUSSION No labeling of i n t a c t c e l l s was observed wherea s labeling occurred with ferritin-SBTI in spermatozoa whose acrosomes were disrupted.
The f e r r i t i n p a r t i c l e s were d i s t r i b u t e d uniformly amid t h e acro-
soma1 contents when such material was s t i l l present between t h e inner and broken outer acrosomal membrane ( f i g . 1 ) . I f the acrosomal contents had dispersed and t h e acrosome was empty, the f e r r i t i n p a r t i c l e s were found on t h e external surface of t h e ruptured outer acrosomal membrane ( f i g . 2 ) . Detached acrosomal caps ( o u t e r acrosomal membranes and associated material ) were a l s o labeled ( f i g . 3 ) .
In no case could f e r r i t i n p a r t i c l e s be observed
on the inner acrosomal membrane ( f i g s . 1-3). The acid treatment removed a l l the f e r r i t i n labeling ( f i g . 4 ) save t h a t seen sometimes on t h e postacrosomal region (vide i n f r a ) .
Free f e r r i t i n d i d
not b i n d t o spermatozoa. Labeling was sometimes observed following ferritin-SBTI incubation on ( b u t not within) t h e equatorial segment of spermatozoa whose a n t e r i o r segment
was disrupted, and, in cases where t h e plasma membrane overlying t h e postacrosomal region had been removed too, f e r r i t i n p a r t i c l e s were a l s o present in the a n t e r i o r half of t h i s region.
Postacrosomal l a b e l i n g , not remcved by
acid treatment, i s probably not due t o acrosin. From these r e s u l t s i t appears, as one might expect, t h a t labeling of acrosin in spermatozoa using ferritin-SBTI can take place only a f t e r t h e acrosome has been disrupted.
I n i n t a c t spermatozoa the intervening plasma
and acrosomal membranes prevent access of ferritin-SBTI t o t h e acrosomal matrix.
Under the conditions of incubation used in our experiments, and 145
from the r e s u l t s of Brown and Harrison ( '78) , i t would appear t h a t , a f t e r acrosomal disruption, the proacrosin within becomes activated t o acrosin, a n d t h i s a c t i v e enzyme i s released i n t o the external s a l i n e milieu.
Effec-
t i v e l y , when material i s s t i l l within t h e disrupted acrosome, f e r r i t i n label appears t o d i f f u s e t h r o u g h the broken outer acrosomal membrane.
Later s t i l l
in the process, label i s found on detached acrosomal caps a n d on the outside of the equatorial segment. As proacrosin has been shown t o a c t i v a t e very r e a d i l y in a bound s t a t e
(Brown a n d Harrison, ' 7 8 ) , one might expect considerable amounts of acrosin t o remain bound t o t h e inner acrosomal membrane and r e a c t t h e r e with f e r r i t i n SBTI i f t h i s were the location of proacrosin.
Although SBTI may r e a c t l e s s
strongly with bound acrosin t h a n with soluble acrosin (Brown and Hartree, ' 7 6 ; Harrison, personal communication) , f e r r i t i n 1 abel was observed on the
material associated with t h e outer acrosomal membrane, suggesting t h a t bound acrosin can indeed be labeled.
Thus t h e absence of labeling on the surface
of the inner acrosomal membrane a t successive s t e p s of acrosome disruption seems a t r u e negative r e s u l t indicating absence of binding of proacrosin o r acrosin t o the inner acrosomal membrane. Green and Hockaday ( ' 7 8 ) have used ferritin-SBTI on guinea pig
FIGURE LEGENDS
All sections a r e stained with uranyl a c e t a t e and lead c i t r a t e . X 42,000. 1-3 Ferritin-conjugated SBTI incubation following acrosomal disruption. The inner acrosomal membrane (IM) i s n o t labeled. Figure 1, section through the a n t e r i o r segment of acrosome, showing f e r r i t i n d i s t r i b u tion over acrosomal matrix ( M A ) and on both s i d e s o f the outer acrosomal membrane ( O M ) . Figure 2 , labeling i s r e s t r i c t e d t o t h e external surface o f the vesiculated outer membrane of the emptied acrosome. Figure 3 , f e r r i t i n p a r t i c l e s a r e associated with detached acrosomal cap. 4 Sperm treated a s in figures 1-3, with subsequent washing in pH 2 . 4 buffer. The a n t e r i o r segment of acrosome i s not labeled. 146
spermatozoa a f t e r an induced acrosomal r e a c t i o n .
From their observations, i t
appears t h a t before t h e l o s s of th e o u t e r acrosomal membrane the bulk of acrosin i s a l r e a d y not located on the inner acrosomal membrane.
Nevertheless
i t would be necessary t o pursue t h e experiments u n t i l shedding of the acrosome cap, a s i t occurs before penetration of the zona pellucida (Noda and Yanagimachi, ' 7 6 ) , t o s e e i f any a c r o s i n would remain on the former membrane in t h i s s p e c i e s .
As i t does not appear t o be bound t o the inner acrosomal membrane a s previously claimed, the f a t e and eventual mode of a c t i o n of a c r o s i n a t the time of f e r t i l i z a t i o n must remain an open question (Bedford and Cross, ' 7 8 ) . ACKNOWLEDGMENTS
Thanks go t o Doctor R. A. P. Harrison f o r c o r r e c t i o n
of the manuscript and C . Slagmulder f o r p r i n t i n g the p l a t e s . G . Shams-Borhan acknowledges a g r a n t from t h e Centre I n t e r n a t i o n a l des
Etudiants e t S t a g i a i r e s ( P a r i s ) and expresses her g r a t i t u d e t o Pr. H . F r i t z f o r advice and encouragement. LITERATURE CITED Bedford, J . M. 1972 An e l e c t r o n microscopic study of sperm penetration i n t o the r a b b i t egg a f t e r natural mating. Am. J . Anat., 133: 213-254. Bedford, J . M., and N . L . Cross 1978 Normal penetration of r a b b i t spermatozoa through a trypsin-and a c r o s i n - r e s i s t a n t zona pellucida. J . Reprod. Fert., 54: 385-392. Brown, C . R . , and R. A. P. Harrison 1978 The a c t i v a t i o n of proacrosin i n spermatozoa from ram, bull and boar. Biochim. Biophys. Acta, 526: 202-217. Brown, C. R., and E . F. Hartree 1974 D i s t r i b u t i o n of a t r y p s i n - l i k e proteinase i n the ram spermatozoon. J . Reprod. Fert., 36: 195-198. Brown, C . R., and E . F. Hartree 1976 E f f e c t s of a c r o s i n i n h i b i t o r s on the s o l u b l e and membrane-bound forms o f ram a c r o s i n , and a reappraisal of the r o l e of the enzyme i n f e r t i l i z a t i o n . HoppeS e y l e r ' s Z. physiol. Chem., 357: 57-65. Flechon, J . - E . , D. Huneau, C . R. Brown and R. A. P. Harrison 1977 Immunocytochemical l o c a l i z a t i o n of acrosin i n the a n t e r i o r segment of the acrosomes of ram, boar and bull spermatozoa. Ann. Biol. anim. Bioch. Biophys., 17: 749-758. Gaddum-Rosse, P . , and R. J . Blandau 1977 Pro.:eolytic a c t i v i t y of guinea-pig spermatozoa a f t e r induction of the acrosomal r e a c t i o n _ in _ v i t_ r o . Amer. J . Anat., 149: 423-430. 148
Green, D. P. L., and A. R. Hockaday 1978 The histochemical localization of acrosin in guinea-pig sperm after the acrosome reaction. J. Cell Sci., 32: 177-184. Hartree, E. F. 1977 Spermatozoa, eggs and proteinases. Biochem. SOC. Trans., 5: 375-394. Kishida, Y., B. R. Olsen, R. A. Berg and D. J. Prockop 1975 Two improved methods for preparing ferritin-protein conjugates for electron microscopy. J. Cell Biol., 64: 331-339. Meizel, S., and S. K. Mukerji 1976 Biochemical studies of proacrosin and acrosin from hamster cauda epididymal spermatozoa. Biol. Reprod., 14: 444-450. Noda, Y. D., and R. Yanagimachi 1976 Electron microscopic observations o f guinea pig spermatozoa penetrating eggs in vitro. Dev. Growth Diff., 18: 15-23. Shams-Borhan, G., W. D. Schleuning, H. Tschesche and H. Fritz 1976 Occurrence of multiple forms of bull and ram acrosin during proenzyme activation and inhibition of activation by p-nitrophenyl-plguanidinobenzoate. Hoppe-Seyler's Z. Physiol. Chem., 357: 667-671. Schill, W. B., and H. H. Wolff 1974 Ultrastructure of human sperm acrosome and determination of acrosin activity under conditions of semen preservation. Int. J. Fertil., 19: 217-223. Yang, S. L., L. J. D. Zaneveld and G. F. B. Schumacher 1976 Effect of serum proteinase inhibitors on the fertilizing capacity of rabbit spermatozoa. Fertil. Steril., 27: 577-581. Zahler, W . L., and G. A. Doak 1975 Isolation of the outer acrosomal membrane from bull sperm. Biochim. Biophys. Acta, 406: 478-488.
149
RAPID COMMUNICATION
ACROSIN DOES NOT APPEAR TO BE BOUND TO THE INNER ACROSOMAL MEMBRANE OF BULL SPERMATOZOA G A B R I E L E SHAMS-BORHAN, DANIEL HUNEAU A N D JACQUES-EDMOND F L ~ C H O N S t a t i o n c e n t r a l e de Physiologie animale, I.N.R.A. 78350 Jouy-en-Josas, France
ABSTRACT Ferritin-conjugated soybean trypsin i n h i b i t o r was used f o r t h e ul t r a s t r u c t u r a l l o c a l i z a t i o n of acrosin in bull spermatozoa following acrosomal disruption. The f e r r i t i n label was observed i n t h e a n t e r i o r segment of the acrosome in disrupted c e l l s only. Emptied acrosomes were labeled, mostly on the external surface of t h e i r outer membrane. Labeling was a l s o found on the material bound t o detached acrosomal caps. However, a t no time could the f e r r i t i n label be found on t h e inner acrosomal membrane. I t i s concluded t h a t acrosin a c t i v i t y i s n o t present on the inner acrosomal membrane b u t i s l o s t from t h e acrosomal matrix as t h e acrosomal reaction proceeds.
Acrosin ( E C 3-4-21-10), a sperm acrosomal proteinase, has been considered an e s s e n t i a l enzyme for the penetration of the zona pellucida by the spermatozoon during f e r t i l i z a t i o n (Meizel and Mukerji , '76; Yang e t a '76, Hartree, ' 7 7 ) .
I t was suggested by Bedford ( ' 7 2 ) t h a t some zona l y s i n
might remain on t h e inner acrosomal membrane a f t e r the acrosome r e a c t on. Subsequently, biochemical s t u d i e s showed t h a t acrosin could be recovered bound t o t h e sperm head in preparations of disrupted c e l l s (Brown and Hartree, '74; Schill and Wolff, ' 7 4 ) . However, t h e immunocytochemical labeling of acrosin, which i s r e s t r i c t e d t o the a n t e r i o r segment of t h e acrosome i n i n t a c t sperm, was progressively abolished in t h i s location during acrosomal disruption and l o s s o f t h e outer acrosomal membrane (F1 ;chon e t a1 . , ' 7 7 ) .
Moreover Gaddum-Rosse and B1 andau
( ' 7 7 ) reported t h a t spermatozoa which had undergone an acrosome reaction no
143
longer showed g e l a t i n o l y t i c a c t i v i t y , and Brown and Harrison ( '78) demonstrated t h a t acrosin i s readily released from sperm heads during proacrosin activation in a s a l i n e medium.
I n a f u r t h e r attempt t o check i f acrosin can be found on the inner acrosoma1 membrane, we have used a f e r r i t i n - l a b e l e d acrosin i n h i b i t o r on bull
spermatozoa a f t e r acrosomal disruption. MATERIAL AND METHODS
Ejaculated semen from b u l l s of proven f e r t i l i t y
was diluted w i t h 2 volumes of 0.15 M NaC1, 5 mM Hepes a t pH 7.5 and c e n t r i fuged through 1 . 3 M sucrose, 0.15 M NaCl a t 1,400 g f o r 25 minutes a t room temperature.
The sperm p e l l e t , a b o u t 0.2 ml, was resuspended in 3 ml o f
0.15 M NaC1, 0.005 M Hepes a t pH 7.5 t o y i e l d a f i n a l sperm concentration of about 2 x lo8 cells/ml
.
The acrosomes , which had been submitted t o t h i s
osmotic shock were f u r t h e r disrupted by passing the sperm c e l l s repeatedly through a Pasteur p i p e t t e (about 50 times).
The whole procedure was derived
from Zahler and Doak ( ' 7 5 ) .
After disruption, human serum albumin (2-20 mg/ml) was added t o prevent non-specific protein adsorption.
A1 iquots ( 0 . 5 ml) of the suspension were
incubated with 0.25 m l of 0 . 1 M T r i s H C l (pH 7.5) containing soybean trypsin i n h i b i t o r (Merck) t h a t had been conjugated with f e r r i t i n (Serva) using glutaraldehyde, according t o Kishida e t a1 . ( ' 7 5 ) .
The solution of conjugated
i n h i b i t o r (ferritin-SBTI) had an a c t i v i t y towards trypsin of 42 mUhl (using
benzoyl-L-arginine-p-nitroanilide as s u b s t r a t e ) and contained between 1 . 2 and 1.7 mg f e r r i t i n / m l .
Control a l i q u o t s were t r e a t e d with 0.15 M formate
buffer pH 2 . 4 a f t e r incubation in f e r r i t i n SBTI t o dissolve t h e acrosin (Shams-Borhan e t a l . , ' 7 6 ) .
Other a l i q u o t s were incubated in equivalent
amounts of unconjugated f e r r i t i n .
All samples were then washed twice with
0.15 m NaC1, 0.005 M Hepes a t pH 7.5, fixed with 1%glutaraldehyde a n d 1% 144
osmium t e t r o x i d e in 0.175 M cacodylate buffer and embedded in epon.
Thin
sections were observed e i t h e r unstained o r a f t e r uranyle a c e t a t e and lead c i t r a t e staining. RESULTS AND DISCUSSION No labeling of i n t a c t c e l l s was observed wherea s labeling occurred with ferritin-SBTI in spermatozoa whose acrosomes were disrupted.
The f e r r i t i n p a r t i c l e s were d i s t r i b u t e d uniformly amid t h e acro-
soma1 contents when such material was s t i l l present between t h e inner and broken outer acrosomal membrane ( f i g . 1 ) . I f the acrosomal contents had dispersed and t h e acrosome was empty, the f e r r i t i n p a r t i c l e s were found on t h e external surface of t h e ruptured outer acrosomal membrane ( f i g . 2 ) . Detached acrosomal caps ( o u t e r acrosomal membranes and associated material ) were a l s o labeled ( f i g . 3 ) .
In no case could f e r r i t i n p a r t i c l e s be observed
on the inner acrosomal membrane ( f i g s . 1-3). The acid treatment removed a l l the f e r r i t i n labeling ( f i g . 4 ) save t h a t seen sometimes on t h e postacrosomal region (vide i n f r a ) .
Free f e r r i t i n d i d
not b i n d t o spermatozoa. Labeling was sometimes observed following ferritin-SBTI incubation on ( b u t not within) t h e equatorial segment of spermatozoa whose a n t e r i o r segment
was disrupted, and, in cases where t h e plasma membrane overlying t h e postacrosomal region had been removed too, f e r r i t i n p a r t i c l e s were a l s o present in the a n t e r i o r half of t h i s region.
Postacrosomal l a b e l i n g , not remcved by
acid treatment, i s probably not due t o acrosin. From these r e s u l t s i t appears, as one might expect, t h a t labeling of acrosin in spermatozoa using ferritin-SBTI can take place only a f t e r t h e acrosome has been disrupted.
I n i n t a c t spermatozoa the intervening plasma
and acrosomal membranes prevent access of ferritin-SBTI t o t h e acrosomal matrix.
Under the conditions of incubation used in our experiments, and 145
from the r e s u l t s of Brown and Harrison ( '78) , i t would appear t h a t , a f t e r acrosomal disruption, the proacrosin within becomes activated t o acrosin, a n d t h i s a c t i v e enzyme i s released i n t o the external s a l i n e milieu.
Effec-
t i v e l y , when material i s s t i l l within t h e disrupted acrosome, f e r r i t i n label appears t o d i f f u s e t h r o u g h the broken outer acrosomal membrane.
Later s t i l l
in the process, label i s found on detached acrosomal caps a n d on the outside of the equatorial segment. As proacrosin has been shown t o a c t i v a t e very r e a d i l y in a bound s t a t e
(Brown a n d Harrison, ' 7 8 ) , one might expect considerable amounts of acrosin t o remain bound t o t h e inner acrosomal membrane and r e a c t t h e r e with f e r r i t i n SBTI i f t h i s were the location of proacrosin.
Although SBTI may r e a c t l e s s
strongly with bound acrosin t h a n with soluble acrosin (Brown and Hartree, ' 7 6 ; Harrison, personal communication) , f e r r i t i n 1 abel was observed on the
material associated with t h e outer acrosomal membrane, suggesting t h a t bound acrosin can indeed be labeled.
Thus t h e absence of labeling on the surface
of the inner acrosomal membrane a t successive s t e p s of acrosome disruption seems a t r u e negative r e s u l t indicating absence of binding of proacrosin o r acrosin t o the inner acrosomal membrane. Green and Hockaday ( ' 7 8 ) have used ferritin-SBTI on guinea pig
FIGURE LEGENDS
All sections a r e stained with uranyl a c e t a t e and lead c i t r a t e . X 42,000. 1-3 Ferritin-conjugated SBTI incubation following acrosomal disruption. The inner acrosomal membrane (IM) i s n o t labeled. Figure 1, section through the a n t e r i o r segment of acrosome, showing f e r r i t i n d i s t r i b u tion over acrosomal matrix ( M A ) and on both s i d e s o f the outer acrosomal membrane ( O M ) . Figure 2 , labeling i s r e s t r i c t e d t o t h e external surface o f the vesiculated outer membrane of the emptied acrosome. Figure 3 , f e r r i t i n p a r t i c l e s a r e associated with detached acrosomal cap. 4 Sperm treated a s in figures 1-3, with subsequent washing in pH 2 . 4 buffer. The a n t e r i o r segment of acrosome i s not labeled. 146
spermatozoa a f t e r an induced acrosomal r e a c t i o n .
From their observations, i t
appears t h a t before t h e l o s s of th e o u t e r acrosomal membrane the bulk of acrosin i s a l r e a d y not located on the inner acrosomal membrane.
Nevertheless
i t would be necessary t o pursue t h e experiments u n t i l shedding of the acrosome cap, a s i t occurs before penetration of the zona pellucida (Noda and Yanagimachi, ' 7 6 ) , t o s e e i f any a c r o s i n would remain on the former membrane in t h i s s p e c i e s .
As i t does not appear t o be bound t o the inner acrosomal membrane a s previously claimed, the f a t e and eventual mode of a c t i o n of a c r o s i n a t the time of f e r t i l i z a t i o n must remain an open question (Bedford and Cross, ' 7 8 ) . ACKNOWLEDGMENTS
Thanks go t o Doctor R. A. P. Harrison f o r c o r r e c t i o n
of the manuscript and C . Slagmulder f o r p r i n t i n g the p l a t e s . G . Shams-Borhan acknowledges a g r a n t from t h e Centre I n t e r n a t i o n a l des
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