RAPID COMMUNICATION
ZIPGRAM
IMMUNOFLUORESCENT LOCALIZATION OF BOVINE ACROSIN ( 1 )
DUANE L. GARNER, MARLYS P. EASTON, MARK E. MUNSON AND MARK A . DOANE Department of Physiological Sciences, Oklahoma S t a t e lniversity, S t i l l w a t e r , Oklahoma 74074 ABSTRACT Acrosin, a t r y p s i n - l i k e proteinase, was localized in t h e acrosome of methanol-fixed bovine spermatozoa by the i n d i r e c t immunofluorescent technique. Anti-acrosin immunoglobulin was obtained from a r a b b i t immunized with highly purified bovine acrosin. The r e s u l t s of agarose-gel immunodiffusion analyses indicated t h a t a1 though the anti body was s p e c i f i c f o r t h e acrosomal enzyme, i t cross-reacted with ovine acrosin. The t r y p s i n - l i k e proteinase, acrosin (Zaneveld, Robertson and Williams, '70; Polakoski e t a l . , ' 7 1 ) , has been demonstrated in the acrosomal materials extracted from bovine spermatozoa (Garner e t a1 . , ' 71 ; Mu1 tamaki and Niemi , ' 7 2 ) . Mu1 tamaki ( ' 7 3 ) a l s o showed t h a t r e l a t i v e l y pure acrosomes i s o l a t e d from bovine spermatozoa
by s u b c e l l u l a r fractionation possessed substantial trypsin-1 i ke a c t i v i t y .
The
apparent importance of acrosin in the f e r t i l i z a t i o n process and the established a n t i g e n i c i t y of bovine acrosomal materials (Zanevel d e t a1 . , ' 74) prompted us t o verify h i s t o l o g i c a l l y the location of acrosin i n the spermatozoan acrosome using i n d i r e c t immunofluorescent s t a i n i n g techniques. MATERIAL AND METHODS Bovine acrosin was p a r t i a l l y purified by a f f i n i t y chromatography (Garner and Cull ison, ' 74) and f u r t h e r purified by preparative electrophoresis (Garner, unpublished) before lyophilization.
The highly purified
acrosin was t r e a t e d with N-a-tosyl -L-lysine chloromethyl ketone HC1 ( T L C K ) before being used f o r immunization.
The TLCK (1 mg) was solublized with a small amount
of dimethyl sulfoxide before d i l u t i o n t o 1 ml with 0.9% NaC1.
The highly purified
acrosin was dissolved in 1 ml o f saline-TLCK before being mixed with an equal 127
volume of Freund's complete adjuvant.
This emulsion was used t o immunize a
mature female New Zealand rabbit using a regimen s i m i l a r t o t h a t outlined by Harboe and Ingild ( ' 7 3 ) . The i n d i r e c t immunofluorescent staining method of Hjort and Hansen ( ' 7 1 ) was used t o l o c a l i z e acrosin.
Krebs-Ringer phosphate buffer (pH 7 . 0 ) ( K R P )
(Umbreit e t a l . , '72) was s u b s t i t u t e d f o r the phosphate buffered s a l i n e .
Washed
spermatozoa were resuspended in KRP and a i r dried before being fixed in absolute methanol f o r 30 min.
The s l i d e s were rinsed with KRP before a drop of a n t i -
acrosin serum was added, and then incubated in a moist chamber f o r 1 hr before being washed twice with KRP.
They were t h e n incubated f o r 30 min. with Fluores-
cein isothiocyanate (F1TC)-conjugated goat a n t i - r a b b i t 7s y-globulin (Hyland) and washed twice before mounting i n a mixture of KRP and glycerol ( 1 : l ) .
Exam-
inations of t h e s l i d e s were made with a Zeiss fluorescence microscope equipped with a HBO 200 w/4 lamp, a UG-5 e x c i t e r f i l t e r and a b a r r i e r f i l t e r t h a t transmits wavelengths above 410 nm. Agarose-gel immunodiffusion p l a t e s (Mi cro-Ouchterlony; Marine Colloids, Inc.,) were used f o r the double immunodiffusion study.
After formation of pre-
c i p i t a t e l i n e s , duplicate plates were stained f o r protein with Coomassie Blue R250 a n d f o r acrosin a c t i v i t y with the chromogenic s u b s t r a t e , N-a-benzoyl-D,
L-arginine-pnaphthylamide HC1 (BANA) (Garner e t a l . , '71) and the coup1 ing-dye, Fast Black K s a l t (ICN - K&K RESULTS
Laboratories).
The acrosomal l o c a l i z a t i o n of bovine acrosin was v e r i f i e d .
As
seen in figure l a , the e n t i r e acrosome possessed a bright fluorescence with a f a i r l y d i s t i n c t posterior l i m i t a t i o n . equatorial segment.
This stained area appeared t o include the
Some spermatozoa which had undergone senescent changes pos-
sessed markedly swollen acrosomes as indicated by the d i f f u s e fluorescent staining "bubble" on the rostra1 portion of the spermatozoan nucleus ( f i g . l b ) . 128
Examination o f t h e s t a i n e d agarose-gel immunodiffusion p l a t e s i n d i c a t e d t h a t t h e antiserum was s p e c i f i c f o r a c r o s i n because i d e n t i c a l p r e c i p i t a t i o n l i n e s were n o t e d f o r t h e p l a t e s s t a i n e d f o r p r o t e i n ( f i g . a c t i v i t y ( f i g . 2b).
2a) o r f o r a c r o s i n
The BANA-amidohydrolase s t a i n e d p l a t e ( f i g . 2b) v e r i f i e d
t h a t t h e a n t i b o d y was a g a i n s t t h e acrosomal enzyme r a t h e r t h a n some o t h e r component e x t r a c t e d from t h e spermatozoan.
The a n t i s e r u m d i d n o t r e a c t w i t h b o v i n e
p a n c r e a t i c t r y p s i n , bovine serum, o r o t h e r b o v i n e acrosomal m a t e r i a l s .
Inter-
e s t i n g l y , t h e a n t i b o d y a l s o recognized o v i n e a c r o s i n . DISCUSSION We have demonstrated t h a t h i g h l y p u r i f i e d b o v i n e a c r o s i n i s
a n t i g e n i c and t h a t t h e immunofluorescent s t a i n i n g method i s s u i t a b l e f o r l o c a l iz a t i o n o f t h e enzyme i n t h e spermatozoan acrosome.
Although t h e s u b c e l l u l a r
r e s o l v i n g power o f t h i s method i s somewhat low, i t was e v i d e n t from examination o f s t a i n e d c e l l s t h a t a t l e a s t a p o r t i o n o f t h e a c r o s i n was l o c a t e d i n t h e o u t e r p o r t i o n o f t h e acrosome.
T h i s phenomenon was noted i n spermatozoa t h a t had
undergone senescent changes and a l s o i n t h e f l u o r e s c e n t s t a i n i n g o f detached acrosomes.
Since t h e presence o f a c r o s i n i n t h e o u t e r l a y e r o f t h e acrosome
may be t h e r e s u l t o f i n t r a - a c r o s o m a l degeneration, a d e f i n i t i v e answer must a w a i t p r e c i s e u l t r a s t r u c t u r a l s t u d i e s of spermatozoa possessing normal acrosomal mo r p ho 1o gy .
The a n t i g e n i c i t y o f a c r o s i n i s an i m p o r t a n t c o n s i d e r a t i o n as a p o s s i b l e cause o f i n f e r t i l i t y i n mammals.
P r e v i o u s l y , Menge ( ' 6 7 ) induced temporary i n -
f e r t i l i t y i n c a t t e by i s o - i m m u n i z a t i o n w i t h semen.
When approached from a
f e r t i l i t y c o n t r o l aspect, t h e immunochemical s p e c i f i c i t y o f a c r o s i n suggests t h a t immunologica
i n t e r f e r e n c e w i t h t h e f u n c t i o n o f t h i s enzyme m i g h t have
advantages o v e r c a t a l y t i c s i t e d i r e c t e d i n t e r f e r e n c e .
These advantages a r e
e s p e c i a l l y i m p o r t a n t s i n c e c a t a l y t i c s i t e d i r e c t e d agents would p r o b a b l y i n t e r f e r e w i t h t h e f u n c t i o n s o f o t h e r p r o t e o l y t i c enzymes i n c l u d i n g t r y p s i n 129
and
plasmi n. LITERATURE CITED Garner, D. L. , G. W. S a l i s b u r y and C. N. Graves 1971 E l e c t r o p h o r e t i c f r a c t i o n a t i o n o f b o v i n e acrosomal p r o t e i n s and p r o t e i n a s e . B i o l . Reprod., 4: 93-100. Garner, D. L., and R. F. C u l l i s o n 1974 P a r t i a l p u r i f i c a t i o n o f bovine a c r o s i n by a f f i n i t y chromatography. J . Chromatog. , 92: 445-449. Harboe, N., and A. I n g i l d 1973 Immunization, i s o l a t i o n o f immunog l o b u l i n s , e s t i m a t i o n o f a n t i b o d y t i t r e . I n : Manual o f Q u a n t i t a t i v e Immunoelectrophoresis Methods and A p p l i c a t i o n s . N. A. Axelsen, J. K r b l l and B. Weeke, U n i v e r s i t e t s s o r l a g e t , Oslo. H j o r t , T. , and K. Brogaard Hansen 1971 Immunofluorescent s t u d i e s on human spermatozoa. I . The d e t e c t i o n o f d i f f e r e n t spermatozoa a n t i b o d i e s and t h e i r occurrence i n normal and i n f e r t i l e women. J . Exp. Immunol., 8: 9-23. Menge, A. C. 1967 Induced i n f e r t i l i t y i n c a t t l e by iso-immunization w i t h semen and t e s t i s . J . Reprod. F e r t i l . , 13: 445-456. Multamaki, S. 1973 I s o l a t i o n o f p u r e acrosomes by s u b c e l l u l a r f r a c t i o n a t i o n o f b u l l spermatozoa. I n t . J . F e r t i l . , 18: 193-205. Multamaki, S. , and M. Niemi 1972 T r y p s i n - l i k e p r o t e o l y t i c a c t i v i t y i n an acrosomal e x t r a c t o f b u l l spermatozoa. I n t . J . F e r t i l . , 17: 43-52. Umbreit, W. W., R. tl. B u r r i s and J . F. S t a u f f e r 1973 Flanometric and Biochemical Techniques, 5 t h e d i t i o n , p. 145, Burgess P u b l i s h i n g Co. , M i nneapol i s . REFERENCE
1 T h i s research was supported, i n p a r t , by a g r a n t (HD 07481) from t h e N a t i o n a l I n s t i t u t e s o f Heal t h y Department o f H e a l t h , Education and We1 f a r e .
FIGURE LEGENDS
1
2
Photomicrographs showing t h e immunofl uorescent r e a c t i o n o f xed bovine spermatozoa. Acrosi n was demonstrated methanol -fi u s i n g t h e i n d i r e c t immunofluorescent technique. Note t h e f l u o r e s c e n c e o f t h e e n t i r e acrosome ( a ) i n d i c a t i n g t h e l o c a t i o n o f a c r o s i n . A l s o n o t e t h e markedly s w o l l e n acrosomes ( b y arrows) o f spermatozoa t h a t had a p p a r e n t l y undergone senescent changes. M a g n i f i c a t i o n X 950. Agarose immunodiffusion p l a t e s o f b o v i n e acrosomal e x t r a c t ( 1 ) , h i g h l y p u r i f i e d b o v i n e a c r o s i n ( 2 ) , o v i n e acrosomal e x t r a c t ( 3 ) , b o v i n e acrosomal e x t r a c t a f t e r a c r o s i n removal ( 4 ) , bovine serum ( 5 ) , and bovine p a n c r e a t i c t r y p s i n ( 6 ) vs. r a b b i t a n t i serum (aA) r a i s e d a g a i n s t b o v i n e a c r o s i n . The p l a t e on t h e l e f t ( a ) was s t a i n e d f o r p r o t e i n and t h e p l a t e on t h e r i g h t ( b ) was s t a i n e d f o r a c r o s i n a c t i v i t y (BANA-amidohydrolase). 130
ZIPGRAM
IMMUNOFLUORESCENT LOCALIZATION OF BOVINE ACROSIN ( 1 )
DUANE L. GARNER, MARLYS P. EASTON, MARK E. MUNSON AND MARK A . DOANE Department of Physiological Sciences, Oklahoma S t a t e lniversity, S t i l l w a t e r , Oklahoma 74074 ABSTRACT Acrosin, a t r y p s i n - l i k e proteinase, was localized in t h e acrosome of methanol-fixed bovine spermatozoa by the i n d i r e c t immunofluorescent technique. Anti-acrosin immunoglobulin was obtained from a r a b b i t immunized with highly purified bovine acrosin. The r e s u l t s of agarose-gel immunodiffusion analyses indicated t h a t a1 though the anti body was s p e c i f i c f o r t h e acrosomal enzyme, i t cross-reacted with ovine acrosin. The t r y p s i n - l i k e proteinase, acrosin (Zaneveld, Robertson and Williams, '70; Polakoski e t a l . , ' 7 1 ) , has been demonstrated in the acrosomal materials extracted from bovine spermatozoa (Garner e t a1 . , ' 71 ; Mu1 tamaki and Niemi , ' 7 2 ) . Mu1 tamaki ( ' 7 3 ) a l s o showed t h a t r e l a t i v e l y pure acrosomes i s o l a t e d from bovine spermatozoa
by s u b c e l l u l a r fractionation possessed substantial trypsin-1 i ke a c t i v i t y .
The
apparent importance of acrosin in the f e r t i l i z a t i o n process and the established a n t i g e n i c i t y of bovine acrosomal materials (Zanevel d e t a1 . , ' 74) prompted us t o verify h i s t o l o g i c a l l y the location of acrosin i n the spermatozoan acrosome using i n d i r e c t immunofluorescent s t a i n i n g techniques. MATERIAL AND METHODS Bovine acrosin was p a r t i a l l y purified by a f f i n i t y chromatography (Garner and Cull ison, ' 74) and f u r t h e r purified by preparative electrophoresis (Garner, unpublished) before lyophilization.
The highly purified
acrosin was t r e a t e d with N-a-tosyl -L-lysine chloromethyl ketone HC1 ( T L C K ) before being used f o r immunization.
The TLCK (1 mg) was solublized with a small amount
of dimethyl sulfoxide before d i l u t i o n t o 1 ml with 0.9% NaC1.
The highly purified
acrosin was dissolved in 1 ml o f saline-TLCK before being mixed with an equal 127
volume of Freund's complete adjuvant.
This emulsion was used t o immunize a
mature female New Zealand rabbit using a regimen s i m i l a r t o t h a t outlined by Harboe and Ingild ( ' 7 3 ) . The i n d i r e c t immunofluorescent staining method of Hjort and Hansen ( ' 7 1 ) was used t o l o c a l i z e acrosin.
Krebs-Ringer phosphate buffer (pH 7 . 0 ) ( K R P )
(Umbreit e t a l . , '72) was s u b s t i t u t e d f o r the phosphate buffered s a l i n e .
Washed
spermatozoa were resuspended in KRP and a i r dried before being fixed in absolute methanol f o r 30 min.
The s l i d e s were rinsed with KRP before a drop of a n t i -
acrosin serum was added, and then incubated in a moist chamber f o r 1 hr before being washed twice with KRP.
They were t h e n incubated f o r 30 min. with Fluores-
cein isothiocyanate (F1TC)-conjugated goat a n t i - r a b b i t 7s y-globulin (Hyland) and washed twice before mounting i n a mixture of KRP and glycerol ( 1 : l ) .
Exam-
inations of t h e s l i d e s were made with a Zeiss fluorescence microscope equipped with a HBO 200 w/4 lamp, a UG-5 e x c i t e r f i l t e r and a b a r r i e r f i l t e r t h a t transmits wavelengths above 410 nm. Agarose-gel immunodiffusion p l a t e s (Mi cro-Ouchterlony; Marine Colloids, Inc.,) were used f o r the double immunodiffusion study.
After formation of pre-
c i p i t a t e l i n e s , duplicate plates were stained f o r protein with Coomassie Blue R250 a n d f o r acrosin a c t i v i t y with the chromogenic s u b s t r a t e , N-a-benzoyl-D,
L-arginine-pnaphthylamide HC1 (BANA) (Garner e t a l . , '71) and the coup1 ing-dye, Fast Black K s a l t (ICN - K&K RESULTS
Laboratories).
The acrosomal l o c a l i z a t i o n of bovine acrosin was v e r i f i e d .
As
seen in figure l a , the e n t i r e acrosome possessed a bright fluorescence with a f a i r l y d i s t i n c t posterior l i m i t a t i o n . equatorial segment.
This stained area appeared t o include the
Some spermatozoa which had undergone senescent changes pos-
sessed markedly swollen acrosomes as indicated by the d i f f u s e fluorescent staining "bubble" on the rostra1 portion of the spermatozoan nucleus ( f i g . l b ) . 128
Examination o f t h e s t a i n e d agarose-gel immunodiffusion p l a t e s i n d i c a t e d t h a t t h e antiserum was s p e c i f i c f o r a c r o s i n because i d e n t i c a l p r e c i p i t a t i o n l i n e s were n o t e d f o r t h e p l a t e s s t a i n e d f o r p r o t e i n ( f i g . a c t i v i t y ( f i g . 2b).
2a) o r f o r a c r o s i n
The BANA-amidohydrolase s t a i n e d p l a t e ( f i g . 2b) v e r i f i e d
t h a t t h e a n t i b o d y was a g a i n s t t h e acrosomal enzyme r a t h e r t h a n some o t h e r component e x t r a c t e d from t h e spermatozoan.
The a n t i s e r u m d i d n o t r e a c t w i t h b o v i n e
p a n c r e a t i c t r y p s i n , bovine serum, o r o t h e r b o v i n e acrosomal m a t e r i a l s .
Inter-
e s t i n g l y , t h e a n t i b o d y a l s o recognized o v i n e a c r o s i n . DISCUSSION We have demonstrated t h a t h i g h l y p u r i f i e d b o v i n e a c r o s i n i s
a n t i g e n i c and t h a t t h e immunofluorescent s t a i n i n g method i s s u i t a b l e f o r l o c a l iz a t i o n o f t h e enzyme i n t h e spermatozoan acrosome.
Although t h e s u b c e l l u l a r
r e s o l v i n g power o f t h i s method i s somewhat low, i t was e v i d e n t from examination o f s t a i n e d c e l l s t h a t a t l e a s t a p o r t i o n o f t h e a c r o s i n was l o c a t e d i n t h e o u t e r p o r t i o n o f t h e acrosome.
T h i s phenomenon was noted i n spermatozoa t h a t had
undergone senescent changes and a l s o i n t h e f l u o r e s c e n t s t a i n i n g o f detached acrosomes.
Since t h e presence o f a c r o s i n i n t h e o u t e r l a y e r o f t h e acrosome
may be t h e r e s u l t o f i n t r a - a c r o s o m a l degeneration, a d e f i n i t i v e answer must a w a i t p r e c i s e u l t r a s t r u c t u r a l s t u d i e s of spermatozoa possessing normal acrosomal mo r p ho 1o gy .
The a n t i g e n i c i t y o f a c r o s i n i s an i m p o r t a n t c o n s i d e r a t i o n as a p o s s i b l e cause o f i n f e r t i l i t y i n mammals.
P r e v i o u s l y , Menge ( ' 6 7 ) induced temporary i n -
f e r t i l i t y i n c a t t e by i s o - i m m u n i z a t i o n w i t h semen.
When approached from a
f e r t i l i t y c o n t r o l aspect, t h e immunochemical s p e c i f i c i t y o f a c r o s i n suggests t h a t immunologica
i n t e r f e r e n c e w i t h t h e f u n c t i o n o f t h i s enzyme m i g h t have
advantages o v e r c a t a l y t i c s i t e d i r e c t e d i n t e r f e r e n c e .
These advantages a r e
e s p e c i a l l y i m p o r t a n t s i n c e c a t a l y t i c s i t e d i r e c t e d agents would p r o b a b l y i n t e r f e r e w i t h t h e f u n c t i o n s o f o t h e r p r o t e o l y t i c enzymes i n c l u d i n g t r y p s i n 129
and
plasmi n. LITERATURE CITED Garner, D. L. , G. W. S a l i s b u r y and C. N. Graves 1971 E l e c t r o p h o r e t i c f r a c t i o n a t i o n o f b o v i n e acrosomal p r o t e i n s and p r o t e i n a s e . B i o l . Reprod., 4: 93-100. Garner, D. L., and R. F. C u l l i s o n 1974 P a r t i a l p u r i f i c a t i o n o f bovine a c r o s i n by a f f i n i t y chromatography. J . Chromatog. , 92: 445-449. Harboe, N., and A. I n g i l d 1973 Immunization, i s o l a t i o n o f immunog l o b u l i n s , e s t i m a t i o n o f a n t i b o d y t i t r e . I n : Manual o f Q u a n t i t a t i v e Immunoelectrophoresis Methods and A p p l i c a t i o n s . N. A. Axelsen, J. K r b l l and B. Weeke, U n i v e r s i t e t s s o r l a g e t , Oslo. H j o r t , T. , and K. Brogaard Hansen 1971 Immunofluorescent s t u d i e s on human spermatozoa. I . The d e t e c t i o n o f d i f f e r e n t spermatozoa a n t i b o d i e s and t h e i r occurrence i n normal and i n f e r t i l e women. J . Exp. Immunol., 8: 9-23. Menge, A. C. 1967 Induced i n f e r t i l i t y i n c a t t l e by iso-immunization w i t h semen and t e s t i s . J . Reprod. F e r t i l . , 13: 445-456. Multamaki, S. 1973 I s o l a t i o n o f p u r e acrosomes by s u b c e l l u l a r f r a c t i o n a t i o n o f b u l l spermatozoa. I n t . J . F e r t i l . , 18: 193-205. Multamaki, S. , and M. Niemi 1972 T r y p s i n - l i k e p r o t e o l y t i c a c t i v i t y i n an acrosomal e x t r a c t o f b u l l spermatozoa. I n t . J . F e r t i l . , 17: 43-52. Umbreit, W. W., R. tl. B u r r i s and J . F. S t a u f f e r 1973 Flanometric and Biochemical Techniques, 5 t h e d i t i o n , p. 145, Burgess P u b l i s h i n g Co. , M i nneapol i s . REFERENCE
1 T h i s research was supported, i n p a r t , by a g r a n t (HD 07481) from t h e N a t i o n a l I n s t i t u t e s o f Heal t h y Department o f H e a l t h , Education and We1 f a r e .
FIGURE LEGENDS
1
2
Photomicrographs showing t h e immunofl uorescent r e a c t i o n o f xed bovine spermatozoa. Acrosi n was demonstrated methanol -fi u s i n g t h e i n d i r e c t immunofluorescent technique. Note t h e f l u o r e s c e n c e o f t h e e n t i r e acrosome ( a ) i n d i c a t i n g t h e l o c a t i o n o f a c r o s i n . A l s o n o t e t h e markedly s w o l l e n acrosomes ( b y arrows) o f spermatozoa t h a t had a p p a r e n t l y undergone senescent changes. M a g n i f i c a t i o n X 950. Agarose immunodiffusion p l a t e s o f b o v i n e acrosomal e x t r a c t ( 1 ) , h i g h l y p u r i f i e d b o v i n e a c r o s i n ( 2 ) , o v i n e acrosomal e x t r a c t ( 3 ) , b o v i n e acrosomal e x t r a c t a f t e r a c r o s i n removal ( 4 ) , bovine serum ( 5 ) , and bovine p a n c r e a t i c t r y p s i n ( 6 ) vs. r a b b i t a n t i serum (aA) r a i s e d a g a i n s t b o v i n e a c r o s i n . The p l a t e on t h e l e f t ( a ) was s t a i n e d f o r p r o t e i n and t h e p l a t e on t h e r i g h t ( b ) was s t a i n e d f o r a c r o s i n a c t i v i t y (BANA-amidohydrolase). 130
