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Inconsistent reactivity of an anti-sperm monoclonal antibody and its relationship to sperm capacitation
Journal of Reproductive Immunology, 9 (1986) 67-70
67
Elsevier JRI 00400
Brief communication
Inconsistent reactivity of an anti-sperm monoclonal antibody and its relationship to sperm capacitation Masaru Okabe, Katsuaki Takada, Toshiyuki Adachi, Yasuhiro K o h a m a and Tsutomu M i m u r a * Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1- 6, Suita, Osaka 565, Japan (Accepted for publication 21 November 1985)
An anti-sperm monoclonal antibody was developed from female C57BL/6 mice immunized with epididymal sperm from a syngeneic male mouse. Immunofluorescence staining revealed that the antibody did not react to fresh epididymal sperm but attached to the capacitated sperm found in the peri-vitelline spaces of the ova. When the antibody was applied to sperm incubated in vitro, variously stained sperm were observed. It was presumed that the antigenic site detected by the antibody was hidden in the fresh epididymal sperm and was spread from the acrosomal cap region to the entire head during the capacitation process. Key words: anti-sperm monoclonal antibody, capacitation fertilization in vitro
The authors have for some years been studying factors which affect the sperm capacitation phenomenon (Aonuma et al., 1978, 1982). Since monoclonal antibodies can be useful probes for studying sperm membrane changes during capacitation, we have become interested in producing these for our own studies. Of the many monoclonal antibodies produced in various laboratories, some have been found to have an inconsistent immunoreactivity to sperm (SMA-2, Feuchter et al., 1984; HS4, Wolf et al., 1983; 8C10.5, Naz et al., 1984; A.F.1, O'Rand et al., 1984). The causes were not stated clearly, however, with the exception of those explained by the tissue- and stage-specific differentiation antigens. We have also experienced the diverse reactivity of a monoclonal antibody and have studied the inconsistency of the immunoreactivity from the viewpoint of sperm surface alteration during capaciration. The clone OBF13 was one of three established from a single fusion procedure. Since it was cloned 5 times by the limiting dilution method and remained unchanged during 10 months of continuous cultivation, we considered the OBF13 antibody as a monoclonal antibody. When OBF13 was incubated with fresh epididymal sperm, few (approximately 5-10%) gave a reaction with the antibody. However, the ratio of positively reacting sperm increased during incubation in vitro. * To whom correspondence should be addressed. 0165-0378/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
68
Fig. 1. Immunofluorescent staining patterns of mouse sperm by OBF13. Monoclonal antibody. OBF13 was developed from female C57BL/6 mice immunized with syngeneic epididymal sperm (intraperitoneal injection of 106 sperm on days 0, 21, 28: spleens were excised on day 32 and fused with myeloma P3U1 using polyethylene glycol). Indirect immunofluorescent staining was employed to detect the antibody binding area. Sperm (approximately 1 × 105) were incubated with 50 /~1 of hybridoma culture supernatant (lst antibody) and 20 btl of FITC-conjugated anti-mouse Ig(A + G + M) (2nd antibody purchased from Miles-Yeda Ltd. and diluted 120 times with PBS supplemented with 5% new-born calf serum (NBCS-PBS)) for 15 min. Sperm were washed with NBCS-PBS prior to each incubation. S i m u l t a n e o u s l y , as shown in Fig. 1, it was o b s e r v e d that the area to which the a n t i b o d y a d h e r e d c h a n g e d (the entire h e a d was stained in a b o u t 30% of the s p e r m after 90 m i n incubation). T h e increase in the stained a r e a was c o m p l e t e l y d e p e n d e n t o n the t e m p e r a t u r e a n d never o c c u r r e d d u r i n g i n c u b a t i o n b e l o w 20°C. T h e reactivity of the a n t i b o d y was d r a s t i c a l l y i n c r e a s e d b y freezing a n d t h a w i n g of the sperm: m o r e t h a n 95% s h o w e d a positive r e a c t i o n to the a n t i b o d y . However, it s e e m e d that the p r o c e d u r e i n c r e a s e d the s t a i n e d s p e r m o n l y in the a c r o s o m a l c a p region. Therefore, it was a s s u m e d that the differently r e a c t i n g s p e r m s h o w n in Fig. 1 r e p r e s e n t e d a c h a n g e in the s p e r m surface d u r i n g c a p a c i t a t i o n . In o r d e r to clarify the r e l a t i o n s h i p b e t w e e n the s t a i n e d a r e a a n d s p e r m c a p a c i t a t i o n we p e r f o r m e d in vitro fertilization o f m o u s e ova ( A o n u m a et al., 1978) a n d tried to stain s p e r m in the peri-vitelline spaces of the ova. O B F 1 3 a t t a c h e d to the entire h e a d of such s p e r m w i t h o u t e x c e p t i o n (Fig. 2). T h e i n t e r a c t i o n of O B F 1 3 with s p e r m was a specific one since n o similar i n t e r a c t i o n was o b s e r v e d when o t h e r m o n o c l o n a l a n t i b o d i e s o r n o r m a l s e r u m were
69
Fig. 2. Sperm in peri-vitelline space stained with OBF13. Fertilized ova collected in less than 1 ktl of medium were introduced to a 10 /~1 droplet of the 1st antibody prepared under paraffin oil and were mixed throughly. After 15 rain, ova were washed with the medium for fertilization and then transferred to 5 ~1 of the 2nd antibody. The entire head was stained in all of the sperm found in the peri-vitelline spaces.
u s e d as a control. W e d o n o t have direct p r o o f t h a t the a n t i g e n r e c o g n i z e d b y O B F 1 3 was on the s p e r m surface. However, s o m e of the s p e r m f o u n d in the peri-vitelline space were still m o t i l e after b e i n g stained. W e therefore c o n s i d e r t h a t the a n t i g e n is p r o b a b l y on the surface. I n Fig. 1 we suggested that O B F 1 3 r e a c t e d with the a c r o s o m a l c a p region, w h i c h we t e n t a t i v e l y i d e n t i f i e d b y the shape of the stained area. However, it was a c t u a l l y difficult to c o n c l u d e w h e t h e r the a r e a o b s e r v e d was the surface or the i n t e r i o r of the a c r o s o m a l cap, o r w h e t h e r it was the newly e x p o s e d surface of the m e m b r a n e after the s h e d d i n g of the a c r o s o m a l cap. It is therefore i m p o r t a n t to follow u p these o b s e r v a t i o n s with further e x p e r i m e n t s utilizing an electron m i c r o s c o p e in o r d e r to d e t e r m i n e the precise l o c a t i o n o f the antigen. This i n f o r m a t i o n w o u l d then help to c l a r i f y the m e m b r a n e changes d u r i n g s p e r m c a p a c i t a t i o n . It is suggested t h a t m o n o c l o n a l a n t i b o d i e s which show weak reactivity to fresh e p i d i d y m a l s p e r m s h o u l d be extensively screened for their reactivity to p e n e t r a t e d s p e r m in o r d e r to ensure r e c o g n i t i o n of p o t e n t i a l l y v a l u a b l e m o n o c l o n a l antibodies.
Acknowledgements W e are grateful to Ms. S a c h i k o T a k a i a n d Ms. K i y o m i N a k a n i s h i for their technical assistance a n d Ms. S t e p h a n i e L. C o o k for her assistance in p r e p a r i n g the manuscript.
References Aonuma, S., Okabe, M. and Kawaguchi, M. (1978) The effect of zinc ions on fertilization of mouse ova in vitro. J. Reprod. Fertil. 53, 179-183. Aonuma, S., Okabe, M., Kishi, Y., Kawaguchi, M. and Yamada, H. (1980) Capacitation inducing activity of serum albumin in fertilization of mouse ova in vitro. J. Pharm. Dyn., 5, 980-987. Feuchter, F.A., Vernon, R.B. and Eddy, E.M. (1981) Analysis of the sperm surface with monoclonal antibodies; topographically restricted antigens appearing in the epididymis. Biol. Reprod. 24, 1099-1110.
70 Naz, R.K., Rosenblum, B.B. and Menge, A.C. (1984) Characterization of a membrane antigen from rabbit testis and sperm isolated by using monoclonal antibodies and effect of its antisperm activity on fertifity. Proc. Nat. Acad. Sci. U.S.A. 81. 857-861. O'Rand. M.G.. Irons, G.P. and Porter, J.P. (1984) Monoclonal antibodies to rabbit sperm autoantigens. 1. Inhibition of in vitro fertilization and localization on the egg. Biol. Reprod. 30. 721-729. Wolf. D.P.. Sokolski. LE., Dandeker, P. and Bechtol, K.B. (1983) Characterization of human sperm surface antigens with monoclonal antibodies. Biol. Reprod, 29. 713-723.
67
Elsevier JRI 00400
Brief communication
Inconsistent reactivity of an anti-sperm monoclonal antibody and its relationship to sperm capacitation Masaru Okabe, Katsuaki Takada, Toshiyuki Adachi, Yasuhiro K o h a m a and Tsutomu M i m u r a * Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1- 6, Suita, Osaka 565, Japan (Accepted for publication 21 November 1985)
An anti-sperm monoclonal antibody was developed from female C57BL/6 mice immunized with epididymal sperm from a syngeneic male mouse. Immunofluorescence staining revealed that the antibody did not react to fresh epididymal sperm but attached to the capacitated sperm found in the peri-vitelline spaces of the ova. When the antibody was applied to sperm incubated in vitro, variously stained sperm were observed. It was presumed that the antigenic site detected by the antibody was hidden in the fresh epididymal sperm and was spread from the acrosomal cap region to the entire head during the capacitation process. Key words: anti-sperm monoclonal antibody, capacitation fertilization in vitro
The authors have for some years been studying factors which affect the sperm capacitation phenomenon (Aonuma et al., 1978, 1982). Since monoclonal antibodies can be useful probes for studying sperm membrane changes during capacitation, we have become interested in producing these for our own studies. Of the many monoclonal antibodies produced in various laboratories, some have been found to have an inconsistent immunoreactivity to sperm (SMA-2, Feuchter et al., 1984; HS4, Wolf et al., 1983; 8C10.5, Naz et al., 1984; A.F.1, O'Rand et al., 1984). The causes were not stated clearly, however, with the exception of those explained by the tissue- and stage-specific differentiation antigens. We have also experienced the diverse reactivity of a monoclonal antibody and have studied the inconsistency of the immunoreactivity from the viewpoint of sperm surface alteration during capaciration. The clone OBF13 was one of three established from a single fusion procedure. Since it was cloned 5 times by the limiting dilution method and remained unchanged during 10 months of continuous cultivation, we considered the OBF13 antibody as a monoclonal antibody. When OBF13 was incubated with fresh epididymal sperm, few (approximately 5-10%) gave a reaction with the antibody. However, the ratio of positively reacting sperm increased during incubation in vitro. * To whom correspondence should be addressed. 0165-0378/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
68
Fig. 1. Immunofluorescent staining patterns of mouse sperm by OBF13. Monoclonal antibody. OBF13 was developed from female C57BL/6 mice immunized with syngeneic epididymal sperm (intraperitoneal injection of 106 sperm on days 0, 21, 28: spleens were excised on day 32 and fused with myeloma P3U1 using polyethylene glycol). Indirect immunofluorescent staining was employed to detect the antibody binding area. Sperm (approximately 1 × 105) were incubated with 50 /~1 of hybridoma culture supernatant (lst antibody) and 20 btl of FITC-conjugated anti-mouse Ig(A + G + M) (2nd antibody purchased from Miles-Yeda Ltd. and diluted 120 times with PBS supplemented with 5% new-born calf serum (NBCS-PBS)) for 15 min. Sperm were washed with NBCS-PBS prior to each incubation. S i m u l t a n e o u s l y , as shown in Fig. 1, it was o b s e r v e d that the area to which the a n t i b o d y a d h e r e d c h a n g e d (the entire h e a d was stained in a b o u t 30% of the s p e r m after 90 m i n incubation). T h e increase in the stained a r e a was c o m p l e t e l y d e p e n d e n t o n the t e m p e r a t u r e a n d never o c c u r r e d d u r i n g i n c u b a t i o n b e l o w 20°C. T h e reactivity of the a n t i b o d y was d r a s t i c a l l y i n c r e a s e d b y freezing a n d t h a w i n g of the sperm: m o r e t h a n 95% s h o w e d a positive r e a c t i o n to the a n t i b o d y . However, it s e e m e d that the p r o c e d u r e i n c r e a s e d the s t a i n e d s p e r m o n l y in the a c r o s o m a l c a p region. Therefore, it was a s s u m e d that the differently r e a c t i n g s p e r m s h o w n in Fig. 1 r e p r e s e n t e d a c h a n g e in the s p e r m surface d u r i n g c a p a c i t a t i o n . In o r d e r to clarify the r e l a t i o n s h i p b e t w e e n the s t a i n e d a r e a a n d s p e r m c a p a c i t a t i o n we p e r f o r m e d in vitro fertilization o f m o u s e ova ( A o n u m a et al., 1978) a n d tried to stain s p e r m in the peri-vitelline spaces of the ova. O B F 1 3 a t t a c h e d to the entire h e a d of such s p e r m w i t h o u t e x c e p t i o n (Fig. 2). T h e i n t e r a c t i o n of O B F 1 3 with s p e r m was a specific one since n o similar i n t e r a c t i o n was o b s e r v e d when o t h e r m o n o c l o n a l a n t i b o d i e s o r n o r m a l s e r u m were
69
Fig. 2. Sperm in peri-vitelline space stained with OBF13. Fertilized ova collected in less than 1 ktl of medium were introduced to a 10 /~1 droplet of the 1st antibody prepared under paraffin oil and were mixed throughly. After 15 rain, ova were washed with the medium for fertilization and then transferred to 5 ~1 of the 2nd antibody. The entire head was stained in all of the sperm found in the peri-vitelline spaces.
u s e d as a control. W e d o n o t have direct p r o o f t h a t the a n t i g e n r e c o g n i z e d b y O B F 1 3 was on the s p e r m surface. However, s o m e of the s p e r m f o u n d in the peri-vitelline space were still m o t i l e after b e i n g stained. W e therefore c o n s i d e r t h a t the a n t i g e n is p r o b a b l y on the surface. I n Fig. 1 we suggested that O B F 1 3 r e a c t e d with the a c r o s o m a l c a p region, w h i c h we t e n t a t i v e l y i d e n t i f i e d b y the shape of the stained area. However, it was a c t u a l l y difficult to c o n c l u d e w h e t h e r the a r e a o b s e r v e d was the surface or the i n t e r i o r of the a c r o s o m a l cap, o r w h e t h e r it was the newly e x p o s e d surface of the m e m b r a n e after the s h e d d i n g of the a c r o s o m a l cap. It is therefore i m p o r t a n t to follow u p these o b s e r v a t i o n s with further e x p e r i m e n t s utilizing an electron m i c r o s c o p e in o r d e r to d e t e r m i n e the precise l o c a t i o n o f the antigen. This i n f o r m a t i o n w o u l d then help to c l a r i f y the m e m b r a n e changes d u r i n g s p e r m c a p a c i t a t i o n . It is suggested t h a t m o n o c l o n a l a n t i b o d i e s which show weak reactivity to fresh e p i d i d y m a l s p e r m s h o u l d be extensively screened for their reactivity to p e n e t r a t e d s p e r m in o r d e r to ensure r e c o g n i t i o n of p o t e n t i a l l y v a l u a b l e m o n o c l o n a l antibodies.
Acknowledgements W e are grateful to Ms. S a c h i k o T a k a i a n d Ms. K i y o m i N a k a n i s h i for their technical assistance a n d Ms. S t e p h a n i e L. C o o k for her assistance in p r e p a r i n g the manuscript.
References Aonuma, S., Okabe, M. and Kawaguchi, M. (1978) The effect of zinc ions on fertilization of mouse ova in vitro. J. Reprod. Fertil. 53, 179-183. Aonuma, S., Okabe, M., Kishi, Y., Kawaguchi, M. and Yamada, H. (1980) Capacitation inducing activity of serum albumin in fertilization of mouse ova in vitro. J. Pharm. Dyn., 5, 980-987. Feuchter, F.A., Vernon, R.B. and Eddy, E.M. (1981) Analysis of the sperm surface with monoclonal antibodies; topographically restricted antigens appearing in the epididymis. Biol. Reprod. 24, 1099-1110.
70 Naz, R.K., Rosenblum, B.B. and Menge, A.C. (1984) Characterization of a membrane antigen from rabbit testis and sperm isolated by using monoclonal antibodies and effect of its antisperm activity on fertifity. Proc. Nat. Acad. Sci. U.S.A. 81. 857-861. O'Rand. M.G.. Irons, G.P. and Porter, J.P. (1984) Monoclonal antibodies to rabbit sperm autoantigens. 1. Inhibition of in vitro fertilization and localization on the egg. Biol. Reprod. 30. 721-729. Wolf. D.P.. Sokolski. LE., Dandeker, P. and Bechtol, K.B. (1983) Characterization of human sperm surface antigens with monoclonal antibodies. Biol. Reprod, 29. 713-723.