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Reduction in fertility by antiserum against rabbit testicular antigen purified using monoclonal antibody
79
l e e n s a n d PBL. (2) s p l e n i c NK a c t i v i t y was d e p r e s s e d from early to mid pregnancy but increased sharply at late pregnancy, declining thereafter. ( 3 ) PBL s h o w e d d e c r e a s e d NK a c t i v i t y during the entire pregnancy, but increased at day 1 of puerperium and returned to the estrous levels thereafter. (4) When s p l e e n c e l l s of early and late pregnancy were fractionated into lymphocytes without phagocytic or adherent cells, the two groups showed no significant difference of NK a c t i v i t y , suggesting that phagocytic and/or adherent cells might be involved in the regulation of splenic NK a c t i v i t y during pregnancy.
VII. Auto- and Iso-immunization to Antigens of Gametes
GUINEA PIG SPERMATOZOA AUTOANTIBODY EFFECT O N SPERM MIGRATION. Brigitte Marquant Le Guienne Jeanne Leung Tack, Marta De Almeida and Guy A. Voisin C.I.P.I.Ex.(U Z3 INSERM. LA Z89 CNRS. C 16 75571 PARIS Cedex 12, F r a n c e
Ass. C1-Bernard), H6p. St-Antoine,
An i n v e r t e d capillary migration t e s t was used as a functional t e s t of guinea-pig epididymal sperm capacity of migration. The open end of a capillary tube containing dilutions of normal guinea-pig IgG or IgG-antibodies against S, P and T s p e r m a t o z o a autoantigens was i m m e r s e d in a suspension of guinea pig epididymal s p e r m a t o z o a . The migration r a t e was d e t e r m i n e d by counting the s p e r m a t o z o a in the capillary tube a f t e r one hour incubation at 25 ° C. There was no d i t f e r e n e e in the migration r a t e observed with normal guinea pig IgGs and a n t i - P or anti-S IgGs (S and P are two soluble, intra acrosomal autoantigens). Anti-T antibodies (T being a plasmic m e m b r a n e autoantigen accessible to specific antibody action) s t i m u l a t e d two or t h r e e times the s p e r m a t o z o a migration. This e f f e c t was only observed when anti-T IgG were in the capillary tube. The a n t i bodies in the sperm suspension were i n e f f e c t i v e . The F o r m y l - M e t . Leu. Phe. peptide well known as a c h e m o t a c t i e f a c t o r for n e u t r o phils and described as a c h e m o a t t r a c t a n t for bull s p e r m a t o z o a had the s a m e e f f e c t on guinea-pig s p e r m a t o z o a . These results suggest that anti-T autoantibodies behave like a c h e m o a t t r a c t i v e agent and t h e r e are not simply stimulating the mobility of s p e r m a t o z o a .
REDUCTION IN FERTILITY BY ANTISERUM AGAINST RABBIT TESTICULAR ANTIGEN PURIFIED USING MONOCLONAL ANTIBODY RAJESH K. NAZ & A. C. MENGE DEPARTMENT OF OB/GYN, UNIVERSITY OF MICHIGAN MEDICAL SCHOOL ANN ARBOR, MICHIGAN Ascites fluid produced by two hybridomas (8C4.1 & 8CI0.5) derived from the same parent clone inhibited fertility in rabbits. Both monoclonal antibodies (MAb) recognized the same antigen from deoxycholate (DOC) solubilized testis using the SDS-gel/protein blot enzyme immunobinding procedure. An
80 immuno-affinity column was prepared with IgG isolated from 8CI0.5 ascites and coupled to glutaraldehyde-activated agarose gel. A preparation of DOCsolubilized testis was run through the MAb affinity column and fractions eluted using pH in increasing steps (pH8-11.4). A purified antigen was isolated showing a single band of 63 kd in SDS-PAGE. The antigen which was identified as a glyco-protein was immunogenic in mice but not in rabbits. The murine antisperm (a/s) was monospeeific recognizing a single protein molecule in single dimensional and 2D gel/protein blot procedure reacting with specific peptides of differing charge but same molecular weight. The a/s which was sperm/testis specific did not agglutinate nor immobilize sperm but did react in immunofluorescence not only with rabbit sperm but also with human and murine sperm. Artificial insemination of female rabbits with a/s-treated sperm resulted in a significant inhibition of fertility at 9 days p.c. without affecting fertilization rates. The post fertilization antifertility effect of a/s was not due to parthenogenie activation of ova or to polyspermy. Passive transfer (i.p.) of antibodies into mice suggested that the anti-63kd antiserum reduced fetal survival. The immuno-contraceptive potential and immunoinfertility diagnostic values of the antigen are under study. PgE 2 M O D U L A T I N G
EFFECTS
IMMUNE
TO SPERM ANTIGENS
RESPONSE
ON THE CELL-MEDIATED
AND
HUMORAL
A.P.TORNYOV, V.H.VULCHANOV, B.A.VELEV I n s t i t u t e of B i o l o g y a n d I m m u n o l o g y of R e p r o d u c t i o n , A c a d e m y of S c i e n c e s , S o f i a , B u l g a r i a
Bulgarian
The i.[). i n t r o d u c t i o n of P r o s t a g l a n d i n Ep (PgEg) in B a l b / c m i c e delays" the r e j e c t i o n of C 5 7 B L 6 - m i c e - g r a f [ s w h i l e the i n j e c t i o n of I n d o m e t h a c i n u m / t h e p r o s t a g l a n d i n - s y n t h e t h a s e inhibitor/ a c c e l e r a t e s t h i s p r o c e s s . The s a m e t e n d e n c i e s are m a r k e d a l s o in the 2 n d set r e a c t i o n , as w e l l in the c a s e s w i t h t r a n s p l a n t a t i o n of m a l e B a l b / c s k i n on f e m a l e s of t h e same s t r a i n / H - Y g e n e t i c d i f f e r e n c e s / . It is n o t a b l e a l s o t h a t B a l b / c - m i c e w h i c h r e c e i v e PgE~ during their immunization with epidydimal spermatozoa from C 5 7 B L 6 r e j e c t m o r e s l o w l y s k i n g r a f t s f r o m the d o n o r s t r a i n in c o m p a r i s o n w i t h m i c e i n j e c t e d w i t h s p e r m a t o z o a only. D a t a s h o w i n g immunosuppressive a c t i o n of P g E 2 on the p r i m a r y a n d s e c o n d a r y h u m o r a l i m m u n e r e s p o n s e to S R B C or to a l l o g e n i c a n d h e t e r o g e n i c spermatozoa were also obtained. A h y p o t h e s i s for the i m m u n o r e g u l a t o r y r o l e of P g E 2 in the d e t e r m i n a t i o n of n a t u r a l i m m u n o l o g i c a l t o l e r a n c e to s p e r m a n t i g e n s in the g e n i t a l t r a c t is b e i n g p r o p o s e d .
ADJUVA}]TS AND ASPEP~MATOGENE,CIS
SEYMOUR KATSH AND GRACE F. KATSI!. DEPARTMENT OF PHA.~MACOLOGY U~IVERSITY OF COLORADO SCIIOOL OF MEDICINE, DE~ER, COLORADO 80262, U..C.A. A single injection of specific antigen (from homologous or autologous sperm, testis or epididymis) when incorporated in appropriate a~juvant, induces deletion of the seminiferous epithelium in the guinea pi~. ['~ile the Freund adjuvant is effective, it is not acceptable for use in humans. After several hundred trials with oils, emulsifiers and bacterial fractions (c.f. Int. Arch. Allergy & Im~unol., 15:172, 1959; idem, 24:319, 1064; i~ature 212:1486, i ~ 6 6 ; Life Sciences, 201:761, 1977) we now find that the mineral oil component can be
l e e n s a n d PBL. (2) s p l e n i c NK a c t i v i t y was d e p r e s s e d from early to mid pregnancy but increased sharply at late pregnancy, declining thereafter. ( 3 ) PBL s h o w e d d e c r e a s e d NK a c t i v i t y during the entire pregnancy, but increased at day 1 of puerperium and returned to the estrous levels thereafter. (4) When s p l e e n c e l l s of early and late pregnancy were fractionated into lymphocytes without phagocytic or adherent cells, the two groups showed no significant difference of NK a c t i v i t y , suggesting that phagocytic and/or adherent cells might be involved in the regulation of splenic NK a c t i v i t y during pregnancy.
VII. Auto- and Iso-immunization to Antigens of Gametes
GUINEA PIG SPERMATOZOA AUTOANTIBODY EFFECT O N SPERM MIGRATION. Brigitte Marquant Le Guienne Jeanne Leung Tack, Marta De Almeida and Guy A. Voisin C.I.P.I.Ex.(U Z3 INSERM. LA Z89 CNRS. C 16 75571 PARIS Cedex 12, F r a n c e
Ass. C1-Bernard), H6p. St-Antoine,
An i n v e r t e d capillary migration t e s t was used as a functional t e s t of guinea-pig epididymal sperm capacity of migration. The open end of a capillary tube containing dilutions of normal guinea-pig IgG or IgG-antibodies against S, P and T s p e r m a t o z o a autoantigens was i m m e r s e d in a suspension of guinea pig epididymal s p e r m a t o z o a . The migration r a t e was d e t e r m i n e d by counting the s p e r m a t o z o a in the capillary tube a f t e r one hour incubation at 25 ° C. There was no d i t f e r e n e e in the migration r a t e observed with normal guinea pig IgGs and a n t i - P or anti-S IgGs (S and P are two soluble, intra acrosomal autoantigens). Anti-T antibodies (T being a plasmic m e m b r a n e autoantigen accessible to specific antibody action) s t i m u l a t e d two or t h r e e times the s p e r m a t o z o a migration. This e f f e c t was only observed when anti-T IgG were in the capillary tube. The a n t i bodies in the sperm suspension were i n e f f e c t i v e . The F o r m y l - M e t . Leu. Phe. peptide well known as a c h e m o t a c t i e f a c t o r for n e u t r o phils and described as a c h e m o a t t r a c t a n t for bull s p e r m a t o z o a had the s a m e e f f e c t on guinea-pig s p e r m a t o z o a . These results suggest that anti-T autoantibodies behave like a c h e m o a t t r a c t i v e agent and t h e r e are not simply stimulating the mobility of s p e r m a t o z o a .
REDUCTION IN FERTILITY BY ANTISERUM AGAINST RABBIT TESTICULAR ANTIGEN PURIFIED USING MONOCLONAL ANTIBODY RAJESH K. NAZ & A. C. MENGE DEPARTMENT OF OB/GYN, UNIVERSITY OF MICHIGAN MEDICAL SCHOOL ANN ARBOR, MICHIGAN Ascites fluid produced by two hybridomas (8C4.1 & 8CI0.5) derived from the same parent clone inhibited fertility in rabbits. Both monoclonal antibodies (MAb) recognized the same antigen from deoxycholate (DOC) solubilized testis using the SDS-gel/protein blot enzyme immunobinding procedure. An
80 immuno-affinity column was prepared with IgG isolated from 8CI0.5 ascites and coupled to glutaraldehyde-activated agarose gel. A preparation of DOCsolubilized testis was run through the MAb affinity column and fractions eluted using pH in increasing steps (pH8-11.4). A purified antigen was isolated showing a single band of 63 kd in SDS-PAGE. The antigen which was identified as a glyco-protein was immunogenic in mice but not in rabbits. The murine antisperm (a/s) was monospeeific recognizing a single protein molecule in single dimensional and 2D gel/protein blot procedure reacting with specific peptides of differing charge but same molecular weight. The a/s which was sperm/testis specific did not agglutinate nor immobilize sperm but did react in immunofluorescence not only with rabbit sperm but also with human and murine sperm. Artificial insemination of female rabbits with a/s-treated sperm resulted in a significant inhibition of fertility at 9 days p.c. without affecting fertilization rates. The post fertilization antifertility effect of a/s was not due to parthenogenie activation of ova or to polyspermy. Passive transfer (i.p.) of antibodies into mice suggested that the anti-63kd antiserum reduced fetal survival. The immuno-contraceptive potential and immunoinfertility diagnostic values of the antigen are under study. PgE 2 M O D U L A T I N G
EFFECTS
IMMUNE
TO SPERM ANTIGENS
RESPONSE
ON THE CELL-MEDIATED
AND
HUMORAL
A.P.TORNYOV, V.H.VULCHANOV, B.A.VELEV I n s t i t u t e of B i o l o g y a n d I m m u n o l o g y of R e p r o d u c t i o n , A c a d e m y of S c i e n c e s , S o f i a , B u l g a r i a
Bulgarian
The i.[). i n t r o d u c t i o n of P r o s t a g l a n d i n Ep (PgEg) in B a l b / c m i c e delays" the r e j e c t i o n of C 5 7 B L 6 - m i c e - g r a f [ s w h i l e the i n j e c t i o n of I n d o m e t h a c i n u m / t h e p r o s t a g l a n d i n - s y n t h e t h a s e inhibitor/ a c c e l e r a t e s t h i s p r o c e s s . The s a m e t e n d e n c i e s are m a r k e d a l s o in the 2 n d set r e a c t i o n , as w e l l in the c a s e s w i t h t r a n s p l a n t a t i o n of m a l e B a l b / c s k i n on f e m a l e s of t h e same s t r a i n / H - Y g e n e t i c d i f f e r e n c e s / . It is n o t a b l e a l s o t h a t B a l b / c - m i c e w h i c h r e c e i v e PgE~ during their immunization with epidydimal spermatozoa from C 5 7 B L 6 r e j e c t m o r e s l o w l y s k i n g r a f t s f r o m the d o n o r s t r a i n in c o m p a r i s o n w i t h m i c e i n j e c t e d w i t h s p e r m a t o z o a only. D a t a s h o w i n g immunosuppressive a c t i o n of P g E 2 on the p r i m a r y a n d s e c o n d a r y h u m o r a l i m m u n e r e s p o n s e to S R B C or to a l l o g e n i c a n d h e t e r o g e n i c spermatozoa were also obtained. A h y p o t h e s i s for the i m m u n o r e g u l a t o r y r o l e of P g E 2 in the d e t e r m i n a t i o n of n a t u r a l i m m u n o l o g i c a l t o l e r a n c e to s p e r m a n t i g e n s in the g e n i t a l t r a c t is b e i n g p r o p o s e d .
ADJUVA}]TS AND ASPEP~MATOGENE,CIS
SEYMOUR KATSH AND GRACE F. KATSI!. DEPARTMENT OF PHA.~MACOLOGY U~IVERSITY OF COLORADO SCIIOOL OF MEDICINE, DE~ER, COLORADO 80262, U..C.A. A single injection of specific antigen (from homologous or autologous sperm, testis or epididymis) when incorporated in appropriate a~juvant, induces deletion of the seminiferous epithelium in the guinea pi~. ['~ile the Freund adjuvant is effective, it is not acceptable for use in humans. After several hundred trials with oils, emulsifiers and bacterial fractions (c.f. Int. Arch. Allergy & Im~unol., 15:172, 1959; idem, 24:319, 1064; i~ature 212:1486, i ~ 6 6 ; Life Sciences, 201:761, 1977) we now find that the mineral oil component can be