RAPID COMMUNICATION
ZIPGRAM
IMMUNOLOGICAL IMMOBILIZATION OF DETACHED RABBIT SPERM TAILS (7 ) CHARLES B. METZ AND MICHAEL G. O'RAND (2) Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Avenue, Coral Gables, Florida 33134 ABSTRACT Motile rabbit sperm tails that lack heads are immobilized by antibody plus complement. Accordingly, immunological immobilization may not necessarily require the extensive sperm acrosomal damage previously reported in immobilized normal sperm. Spermatozoa are rapidly immobilized when exposed to antisperm antibodies in the presence of complement (C').
By analogy with immunological hemolysis
(Iles et a1 ., ' 73) increased sperm cell membrane permeability and ul trastructurally resolvable damage should be associated with such sperm immobilization. Increased permeability and massive disruption of the cell membrane in the acrosoma1 region have been reported for bull (Drevius, '68) and rabbit sperm (Russo and Metz, ' 74), respectively. The membrane damage to immunologically immobilized rabbit sperm can be resolved by scanning electron microscopy and imnunofluorescence with light optics (Russo, Metz and Dunbar, '75). The membrane damage or lesions in the acrosomal region adequately explains immunological immobi 1 ization of rabbit sperm (Russo and Metz, '74). However, these acrosomal lesions could be a secondary manifestation of more general membrane damage not readily resolved by electron microscopy. Sperm immobilization without sperm head membrane involvement would support this hypothesis and demonstrate that gross membrane lesions in the acrosomal region are not the primary explanation for immunological immobilization. The simplest test for this would be examination for immunological immobilization of 301
t a i l s detached from sperm heads.
We have now demonstrated such i m m o b i l i z a t i o n
o f detached sperm t a i l s . MATERIALS AND METHODS M o t i l e , detached sperm t a i l s were o b t a i n e d from e j a c u l a t e s o f an unusual r a b b i t .
The e j a c u l a t e s c o n s i s t e n t l y c o n t a i n e d approx-
i m a t e l y 50% normal m o t i l e sperm and 50% separate sperm heads and m o t i l e t a i l s . When necessary detached t a i l s were separated from most o f t h e normal sperm, detached heads and seminal p a r t i c l e s (Metr e t a1
. , ' 68)
by c e n t r i f u g i n g (approx.
500 g; 5 min) semen samples a f t e r d i l u t i o n t o 25% w i t h PBS (0.01M phosphate, 0.85% NaCl, pH 7).
Supernatants were r e l a t i v e l y p u r e suspensions o f detached
sperm t a i l s . A n t i s e r a were o b t a i n e d from goats immunized w i t h whole r a b b i t semen i n Freunds a d j u v a n t emulsion (Metz and Anika, controls.
'70).
P r e i n j e c t i o n sera served as
G l o b u l i n s were separated from sera by 18%NapS04 p r e c i p i t a t i o n and
d i a l y s i s a g a i n s t PBS y i e l d i n g approximately 15 mg/ml p r o t e i n . (56'C,
30 min) t o d e s t r o y C ' .
Sera were heated
To t e s t f o r i m m o b i l i z a t i o n 25% semen o r t h e de-
tached sperm t a i l suspension, a n t i b o d y g l o b u l i n and guinea p i g C ' .
(Cordis
L a b o r a t o r i e s , Miami, F l o r i d a ) were mixed i n equal p r o p o r t i o n s (Russo and Metz, ' 7 4 ) and examined w i t h phase o p t i c s .
L i v i n g sperm and detached t a i l s were
photographed u s i n g e l e c t r o n i c f l a s h and Kodak h i g h c o n t r a s t copy f i l m . RESULTS A semen sample c o n t a i n i n g separate sperm heads and t a i l s and normal sperm i s i l l u s t r a t e d i n f i g u r e 1. a l l o f t h e sperm midpiece.
The sperm t a i l s appear t o i n c l u d e most i f n o t
Separate heads appear t o l a c k even a t a i l "stump".
On t h e assumption t h a t separate heads and t a i l s a r i s e by s e p a r a t i o n o f p r e v i o u s l y i n t a c t sperm t h i s s e p a r a t i o n o r detachment must occur a t t h e sperm head
-
midpiece j u n c t i o n producing detached t a i l s w i t h an e s s e n t i a l l y complete l o c o motor apparatus.
Many detached sperm t a i l s were immobile i n f r e s h e j a c u l a t e s .
However, a s i g n i f i c a n t number were a c t i v e . 302
Movement o f these v a r i e d from
s t a t i o n a r y a c t i o n t o r a p i d f o r w a r d progression. body and
c',
Following a d d i t i o n o f a n t i -
sperm and a c t i v e detached t a i l s were i m m o b i l i z e d w i t h i n 10 minutes.
I m m o b i l i z a t i o n was n o t observed i n absence o f a c t i v e C ' o r a n t i b o d y ( t a b l e 1 ) . DISCUSSION
E j a c u l a t e s c o n t a i n i n g s i g n i f i c a n t numbers o f detached, progres-
s i v e l y m o t i l e sperm t a i l s have been r e p o r t e d p r e v i o u s l y (see Nelson, '67). Since t h e detached r a b b i t sperm t a i l s a r e i m m o b i l i z e d by a n t i b o d y p l u s C ' ,
i t i s concluded t h a t sperm heads a r e n o t e s s e n t i a l f o r sperm i m m o b i l i z a t i o n . A c c o r d i n g l y , " i m m o b i l i z i n g a n t i g e n s " a r e n e i t h e r c o n f i n e d t o t h e sperm head n o r arranged t h e r e i n a unique and e s s e n t i a l c o n f i g u r a t i o n .
.This i s c o n s i s t e n t
w i t h a g g l u t i n a t i o n and immunofluorescence s t u d i e s u s i n g a n t i s e r a t o i s o l a t e d sperm membrane g l y c o p r o t e i n s .
These demonstrate an i m m o b i l i z i n g sperm membrane
a n t i g e n (O'Rand and Metz, ' 7 4 ) on b o t h heads and t a i l s o f r a b b i t sperm. F i n a l l y , immunological i m m o b i l i z a t i o n o f r a b b i t sperm i s a s s o c i a t e d w i t h r e l a t i v e l y massive d i s r u p t i o n o f t h e c e l l membrane i n t h e acrosomal r e g i o n (Russo and Metz, ' 7 4 ) .
I t i s c l e a r from t h e f o r g o i n g t h a t t h i s membrane damage
can n o t adequately e x p l a i n immunological i m m o b i l i z a t i o n .
I t may be a secondary
m a n i f e s t a t i o n p o s s i b l y r e s u l t i n g from t h e r e l e a s e o f h y d r o l y t i c acrosomal enzymes f o l l o w i n g t h e a s s o c i a t e d damage t o t h e o u t e r acrosomal membrane. The mechanism o f t h e detached t a i l i m m o b i l i z a t i o n and t h e p r i m a r y cause o f i n t a c t sperm i m m o b i l i z a t i o n remains obscure.
One p o s s i b i l i t y , namely a n t i b o d y
i n h i b i t i o n o f an e s s e n t i a l membrane t r a n s p o r t enzyme(s) seems r u l e d o u t by t h e C ' requirement.
T h i s requ rement i s most e a s i l y e x p l a i n e d
immunological hemolysis i n terms o f c e l l membrane damage.
by analogy w i t h It s t i l l seems
(Russo and Metz, ' 7 4 ) l i k e y, then, t h a t i n c r e a s e d p e r m e a b i l i t y a s s o c i a t e d w i t h membrane damage produces i m m o b i l i z a t i o n .
Even though 10 nm " h o l e s " comparable
t o those i n l y s e d e r y t h r o c y t e membranes were n o t found i n n e g a t i v e l y s t a i n e d "ghosts" from i m m o b i l i z e d sperm (Russo and Metz, '74), f u r t h e r i n v e s t i g a t i o n
303
may r e v e a l these o r comparable evidence o f damage t o t h e sperm c e l l membrane a s s o c i a t e d w i t h immunological i m m o b i l i z a t i o n . ACKNOWLEDGMENTS We a r e i n d e b t e d t o Ms. Bonnie Dunbar and M r . F r e d e r i c k Bartram f o r t e c h n i c a l assistance. LITERATURE C I T E D Drevius, L. 0. 1968 On t h e mechanism o f sperm i m m o b i l i z a t i o n induced by normal sera and antisemen sera. E x p t l . C e l l Research, 51: 362-394. I l e s , G. H., P. Seeman, D. N a y l o r and B. Cinader 1973 Membrane l e s i o n s i n immune l y s i s . J. C e l l B i o l . , 56: 528-539. Metz, C. G., and J. Anika 1970 F a i l u r e o f c o n c e p t i o n i n r a b b i t s i n seminated w i t h n o n a g g l u t i n a t i n g , u n i v a l e n t a n t i b o d y - t r e a t e d semen. B i o l . Reprod., 2: 284-290. Metz, C. B., G. W. Hinsch and J . Anika 1968 U l t r a s t r u c t u r e and a n t i g e n s o f p a r t i c l e s from r a b b i t semen. J. Reprod. F e r t . , 17: 195-198. Nelson, L. 1967 Sperm m o t i l i t y i n : F e r t i l i z a t i o n : Comparative Morphology, B i o c h e m i s t r y and Immunology. C. B. Metz and A. Monray, eds Academic Press, New York. O'Rand, M. G., and C. B. Metz 1974 I s o l a t i o n of sperm s u r f a c e a n t i g e n s by e x t r a c t i o n o f r a b b i t spermatozoan membranes w i t h l i t h i u m d i i o d o s a l i c y l a t e . J. C e l l B i o l . , 63: 252a, a b s t r a c t . Russo, J . , and C. B. Metz 1974 The u l t r a s t r u c t u r a l l e s i o n s induced by a n t i b o d y and complement i n r a b b i t spermatozoa. B i o l . Reprod. , 10: 293-308. Russo, J . , C. B. Metz and B. S. Dunbar 1975 Membrane damage t o immunol o g i c a l l y i m m o b i l i z e d r a b b i t spermatozoa v i s u a l i z e d by immunofluorescence and scanning e l e c t r o n microscopy. s u b m i t t e d t o B i o l . Reprod. REFERENCES
1 C o n t r i b u t i o n 285 from t h e I n s t i t u t e f o r Molecular and C e l l u l a r E v o l u t i o n . The s t u d y was a i d e d by g r a n t s from t h e P o p u l a t i o n Council M74-31 and t h e N a t i o n a l I n s t i t u t e f o r C h i l d H e a l t h and Human Development 1-R01-HD06663-01. 2 Supported by U n i t e d S t a t e s P u b l i c H e a l t h S e r v i c e P o s t d o c t o r a l Fellows h i p N I H 5F02 HD53900-02. FIGURE LEGEND 1
L i v i n g sperm, detached t a i l s , and detached heads i n semen from unusual r a b b i t . T y p i c a l wave p a t t e r n o f m o t i l e sperm t a i l s i s e v i d e n t i n whole sperm and some o f t h e detached t a i l s . Phase o p t i c s ; e l e c t r o n i c f l a s h ; approximately X 430.
304
M o t i l i t y o f d e t a c h e d r a b b i t sperm t a i l s and normal sperm i n g o a t a n t i r a b b i t sperm g l o b u l i n and complement (C') Antisperm g l o b u l i n C'
Heated C '
Control globulin
Saline
C'
Heated C '
Saline
k a c h e d t a i 1s
-
+
+
t
t
t
Irma1 sperm
-
t
t
t
t
t
ZIPGRAM
IMMUNOLOGICAL IMMOBILIZATION OF DETACHED RABBIT SPERM TAILS (7 ) CHARLES B. METZ AND MICHAEL G. O'RAND (2) Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Avenue, Coral Gables, Florida 33134 ABSTRACT Motile rabbit sperm tails that lack heads are immobilized by antibody plus complement. Accordingly, immunological immobilization may not necessarily require the extensive sperm acrosomal damage previously reported in immobilized normal sperm. Spermatozoa are rapidly immobilized when exposed to antisperm antibodies in the presence of complement (C').
By analogy with immunological hemolysis
(Iles et a1 ., ' 73) increased sperm cell membrane permeability and ul trastructurally resolvable damage should be associated with such sperm immobilization. Increased permeability and massive disruption of the cell membrane in the acrosoma1 region have been reported for bull (Drevius, '68) and rabbit sperm (Russo and Metz, ' 74), respectively. The membrane damage to immunologically immobilized rabbit sperm can be resolved by scanning electron microscopy and imnunofluorescence with light optics (Russo, Metz and Dunbar, '75). The membrane damage or lesions in the acrosomal region adequately explains immunological immobi 1 ization of rabbit sperm (Russo and Metz, '74). However, these acrosomal lesions could be a secondary manifestation of more general membrane damage not readily resolved by electron microscopy. Sperm immobilization without sperm head membrane involvement would support this hypothesis and demonstrate that gross membrane lesions in the acrosomal region are not the primary explanation for immunological immobilization. The simplest test for this would be examination for immunological immobilization of 301
t a i l s detached from sperm heads.
We have now demonstrated such i m m o b i l i z a t i o n
o f detached sperm t a i l s . MATERIALS AND METHODS M o t i l e , detached sperm t a i l s were o b t a i n e d from e j a c u l a t e s o f an unusual r a b b i t .
The e j a c u l a t e s c o n s i s t e n t l y c o n t a i n e d approx-
i m a t e l y 50% normal m o t i l e sperm and 50% separate sperm heads and m o t i l e t a i l s . When necessary detached t a i l s were separated from most o f t h e normal sperm, detached heads and seminal p a r t i c l e s (Metr e t a1
. , ' 68)
by c e n t r i f u g i n g (approx.
500 g; 5 min) semen samples a f t e r d i l u t i o n t o 25% w i t h PBS (0.01M phosphate, 0.85% NaCl, pH 7).
Supernatants were r e l a t i v e l y p u r e suspensions o f detached
sperm t a i l s . A n t i s e r a were o b t a i n e d from goats immunized w i t h whole r a b b i t semen i n Freunds a d j u v a n t emulsion (Metz and Anika, controls.
'70).
P r e i n j e c t i o n sera served as
G l o b u l i n s were separated from sera by 18%NapS04 p r e c i p i t a t i o n and
d i a l y s i s a g a i n s t PBS y i e l d i n g approximately 15 mg/ml p r o t e i n . (56'C,
30 min) t o d e s t r o y C ' .
Sera were heated
To t e s t f o r i m m o b i l i z a t i o n 25% semen o r t h e de-
tached sperm t a i l suspension, a n t i b o d y g l o b u l i n and guinea p i g C ' .
(Cordis
L a b o r a t o r i e s , Miami, F l o r i d a ) were mixed i n equal p r o p o r t i o n s (Russo and Metz, ' 7 4 ) and examined w i t h phase o p t i c s .
L i v i n g sperm and detached t a i l s were
photographed u s i n g e l e c t r o n i c f l a s h and Kodak h i g h c o n t r a s t copy f i l m . RESULTS A semen sample c o n t a i n i n g separate sperm heads and t a i l s and normal sperm i s i l l u s t r a t e d i n f i g u r e 1. a l l o f t h e sperm midpiece.
The sperm t a i l s appear t o i n c l u d e most i f n o t
Separate heads appear t o l a c k even a t a i l "stump".
On t h e assumption t h a t separate heads and t a i l s a r i s e by s e p a r a t i o n o f p r e v i o u s l y i n t a c t sperm t h i s s e p a r a t i o n o r detachment must occur a t t h e sperm head
-
midpiece j u n c t i o n producing detached t a i l s w i t h an e s s e n t i a l l y complete l o c o motor apparatus.
Many detached sperm t a i l s were immobile i n f r e s h e j a c u l a t e s .
However, a s i g n i f i c a n t number were a c t i v e . 302
Movement o f these v a r i e d from
s t a t i o n a r y a c t i o n t o r a p i d f o r w a r d progression. body and
c',
Following a d d i t i o n o f a n t i -
sperm and a c t i v e detached t a i l s were i m m o b i l i z e d w i t h i n 10 minutes.
I m m o b i l i z a t i o n was n o t observed i n absence o f a c t i v e C ' o r a n t i b o d y ( t a b l e 1 ) . DISCUSSION
E j a c u l a t e s c o n t a i n i n g s i g n i f i c a n t numbers o f detached, progres-
s i v e l y m o t i l e sperm t a i l s have been r e p o r t e d p r e v i o u s l y (see Nelson, '67). Since t h e detached r a b b i t sperm t a i l s a r e i m m o b i l i z e d by a n t i b o d y p l u s C ' ,
i t i s concluded t h a t sperm heads a r e n o t e s s e n t i a l f o r sperm i m m o b i l i z a t i o n . A c c o r d i n g l y , " i m m o b i l i z i n g a n t i g e n s " a r e n e i t h e r c o n f i n e d t o t h e sperm head n o r arranged t h e r e i n a unique and e s s e n t i a l c o n f i g u r a t i o n .
.This i s c o n s i s t e n t
w i t h a g g l u t i n a t i o n and immunofluorescence s t u d i e s u s i n g a n t i s e r a t o i s o l a t e d sperm membrane g l y c o p r o t e i n s .
These demonstrate an i m m o b i l i z i n g sperm membrane
a n t i g e n (O'Rand and Metz, ' 7 4 ) on b o t h heads and t a i l s o f r a b b i t sperm. F i n a l l y , immunological i m m o b i l i z a t i o n o f r a b b i t sperm i s a s s o c i a t e d w i t h r e l a t i v e l y massive d i s r u p t i o n o f t h e c e l l membrane i n t h e acrosomal r e g i o n (Russo and Metz, ' 7 4 ) .
I t i s c l e a r from t h e f o r g o i n g t h a t t h i s membrane damage
can n o t adequately e x p l a i n immunological i m m o b i l i z a t i o n .
I t may be a secondary
m a n i f e s t a t i o n p o s s i b l y r e s u l t i n g from t h e r e l e a s e o f h y d r o l y t i c acrosomal enzymes f o l l o w i n g t h e a s s o c i a t e d damage t o t h e o u t e r acrosomal membrane. The mechanism o f t h e detached t a i l i m m o b i l i z a t i o n and t h e p r i m a r y cause o f i n t a c t sperm i m m o b i l i z a t i o n remains obscure.
One p o s s i b i l i t y , namely a n t i b o d y
i n h i b i t i o n o f an e s s e n t i a l membrane t r a n s p o r t enzyme(s) seems r u l e d o u t by t h e C ' requirement.
T h i s requ rement i s most e a s i l y e x p l a i n e d
immunological hemolysis i n terms o f c e l l membrane damage.
by analogy w i t h It s t i l l seems
(Russo and Metz, ' 7 4 ) l i k e y, then, t h a t i n c r e a s e d p e r m e a b i l i t y a s s o c i a t e d w i t h membrane damage produces i m m o b i l i z a t i o n .
Even though 10 nm " h o l e s " comparable
t o those i n l y s e d e r y t h r o c y t e membranes were n o t found i n n e g a t i v e l y s t a i n e d "ghosts" from i m m o b i l i z e d sperm (Russo and Metz, '74), f u r t h e r i n v e s t i g a t i o n
303
may r e v e a l these o r comparable evidence o f damage t o t h e sperm c e l l membrane a s s o c i a t e d w i t h immunological i m m o b i l i z a t i o n . ACKNOWLEDGMENTS We a r e i n d e b t e d t o Ms. Bonnie Dunbar and M r . F r e d e r i c k Bartram f o r t e c h n i c a l assistance. LITERATURE C I T E D Drevius, L. 0. 1968 On t h e mechanism o f sperm i m m o b i l i z a t i o n induced by normal sera and antisemen sera. E x p t l . C e l l Research, 51: 362-394. I l e s , G. H., P. Seeman, D. N a y l o r and B. Cinader 1973 Membrane l e s i o n s i n immune l y s i s . J. C e l l B i o l . , 56: 528-539. Metz, C. G., and J. Anika 1970 F a i l u r e o f c o n c e p t i o n i n r a b b i t s i n seminated w i t h n o n a g g l u t i n a t i n g , u n i v a l e n t a n t i b o d y - t r e a t e d semen. B i o l . Reprod., 2: 284-290. Metz, C. B., G. W. Hinsch and J . Anika 1968 U l t r a s t r u c t u r e and a n t i g e n s o f p a r t i c l e s from r a b b i t semen. J. Reprod. F e r t . , 17: 195-198. Nelson, L. 1967 Sperm m o t i l i t y i n : F e r t i l i z a t i o n : Comparative Morphology, B i o c h e m i s t r y and Immunology. C. B. Metz and A. Monray, eds Academic Press, New York. O'Rand, M. G., and C. B. Metz 1974 I s o l a t i o n of sperm s u r f a c e a n t i g e n s by e x t r a c t i o n o f r a b b i t spermatozoan membranes w i t h l i t h i u m d i i o d o s a l i c y l a t e . J. C e l l B i o l . , 63: 252a, a b s t r a c t . Russo, J . , and C. B. Metz 1974 The u l t r a s t r u c t u r a l l e s i o n s induced by a n t i b o d y and complement i n r a b b i t spermatozoa. B i o l . Reprod. , 10: 293-308. Russo, J . , C. B. Metz and B. S. Dunbar 1975 Membrane damage t o immunol o g i c a l l y i m m o b i l i z e d r a b b i t spermatozoa v i s u a l i z e d by immunofluorescence and scanning e l e c t r o n microscopy. s u b m i t t e d t o B i o l . Reprod. REFERENCES
1 C o n t r i b u t i o n 285 from t h e I n s t i t u t e f o r Molecular and C e l l u l a r E v o l u t i o n . The s t u d y was a i d e d by g r a n t s from t h e P o p u l a t i o n Council M74-31 and t h e N a t i o n a l I n s t i t u t e f o r C h i l d H e a l t h and Human Development 1-R01-HD06663-01. 2 Supported by U n i t e d S t a t e s P u b l i c H e a l t h S e r v i c e P o s t d o c t o r a l Fellows h i p N I H 5F02 HD53900-02. FIGURE LEGEND 1
L i v i n g sperm, detached t a i l s , and detached heads i n semen from unusual r a b b i t . T y p i c a l wave p a t t e r n o f m o t i l e sperm t a i l s i s e v i d e n t i n whole sperm and some o f t h e detached t a i l s . Phase o p t i c s ; e l e c t r o n i c f l a s h ; approximately X 430.
304
M o t i l i t y o f d e t a c h e d r a b b i t sperm t a i l s and normal sperm i n g o a t a n t i r a b b i t sperm g l o b u l i n and complement (C') Antisperm g l o b u l i n C'
Heated C '
Control globulin
Saline
C'
Heated C '
Saline
k a c h e d t a i 1s
-
+
+
t
t
t
Irma1 sperm
-
t
t
t
t
t
