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Studies of sperm antigens using specific monoclonal antibodies Sun, Ping 1987

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STUDIES OF SPERM ANTIGENS USING SPECIFIC MONOCLONAL ANTIBODIES BY PING SUN M.B. Beijing Second Medical College 1982 M.Sc. Peking Union Medical College 19S5 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE ln THE FACULTY OF GRADUATE STUDIES Department of Gynecology and O b s t e t r i c s Human Reproductive Biology Programme We accept t h i s t h e s i s as conforming t o the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1987 Q Ping Sun, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Q fV?, .i-g-(-r -"C-c. (S-tjna -o-oo I e ^-^j The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6(3/81) A B S T R A C T T h i s t h e s i s w o r k i s f o c u s e d o n s t u d i e s o f s p e r m a n t i g e n : i t s p u r i f i c a t i o n a n d a n t i f e r t i l i t y e f f e c t , i t s " i n t e r n a l i m a g e " b y u s e o f a n t i - i d i o t y p i c a n t i b o d i e s , a n d i t s c a p a c i t y t o i n d u c e a n t i b o d i e s o f IgA c l a s s . M o u s e s p e r m a c r o s o m a l a n t i g e n s t h a t r e a c t w i t h a m o n o c l o n a l a n t i b o d y g e n e r a t e d a g a i n s t m o u s e s p e r m a t o z o a , MS 2 0 7 , w e r e p u r i f i e d b y i m m u n o a f f i n i t y c h r o m a t o g r a p h y f r o m s o l u b l e f r a c t i o n o f m o u s e t e s t i s h o m o g e n a t e s . A n a l y z e d b y H P L C a n d S D S g e l e l e c t r o p h o r e s i s i n t h e p r e s e n c e o f r e d u c i n g a g e n t , t h e p u r i f i e d s p e r m a n t i g e n s w e r e s h o w n t o h a v e a m o l e c u l a r w e i g h t o f 6 0 0 t o 7 0 0 K D a . It w a s s h o w n t o b e g l y c o p r o t e i n s w i t h c a r b o h y d r a t e c o n t e n t o f 23%. T h e p u r i f i e d m o u s e s p e r m a n t i g e n w a s u s e d t o i s o i m m u n i z e f e m a l e m i c e . A f t e r s u c c e s s i v e i m m u n i z a t i o n s , a n t i s e r a o f h i g h t i t e r s w e r e r a i s e d a n d w e r e s h o w n t o r e a c t s p e c i f i c a l l y w i t h m o u s e a n d h u m a n s p e r m , b u t n o t w i t h a n y m o u s e s o m a t i c t i s s u e s a s j u d g e d f r o m r e s u l t s o f i m m u n o f l u o r e s c e n t a s s a y a n d O u c h t e r l o n y ' s d o u b l e i m m u n o d i f f u s i o n . T h e a n t i s e r a w e r e s h o w n t o c r o s s r e a c t w i t h h u m a n s p e r m a t t h e a c r o s o m a l r e g i o n . B y u s i n g m o u s e i n v i t r o f e r t i l i z a t i o n e x p e r i m e n t s a n d h u m a n s p e r m p e n e t r a t i o n a s s a y w i t h z o n a - f r e e h a m s t e r o v a , t h e a n t i s e r a w e r e s h o w n t o e x e r t s t r o n g i n h i b i t o r y e f f e c t s o n f e r t i l i z a t i o n . - i i -A n o t h e r p a r t o f t h i s t h e s i s w o r k w a s d i r e c t e d t o g e n e r a t i n g a n t i - i d i o t y p i c a n t i b o d y a g a i n s t a m o n o c l o n a l s p e r m a n t i b o d y MS 2 0 4 . MS 2 0 4 w a s p r e v i o u s l y s h o w n t o r e a c t w i t h a c o n f o r m a t i o n a l e p i t o p e o f s p e r m a n t i g e n s . A n t i - M S 2 0 4 a n t i s e r u m w a s r a i s e d i n a r a b b i t . A s e r i e s o f a f f i n i t y c h r o m a t o g r a p h i c c o l u m n s w e r e a p p l i e d t o p u r i f y a n t i - F a b f r a g m e n t o f MS 2 0 4 ( A i d 2 0 4 ) . T h e o b t a i n e d A i d 2 0 4 p o s s e s s e d w e a k a n t i g e n i c i t y i n t h e m o u s e . A f t e r s u c c e s s i v e i m m u n i z a t i o n s w i t h A i d 2 0 4 , t h e i s o i m m u n e s e r a a g a i n s t A i d 2 0 4 w e r e s h o w n t o c r o s s r e a c t w i t h a c r o s o m a l r e g i o n o f m o u s e s p e r m , a p a t t e r n s i m i l a r t o t h a t o f MS 2 0 4 . A p a r t i a l i n h i b i t o r y e f f e c t o n i n v i t r o f e r t i l i z a t i o n w a s o b s e r v e d . T h e r e s u l t s i n d i c a t e t h a t t h e " i n t e r n a l i m a g e " o f t h e s p e c i f i c s p e r m a n t i g e n r e a c t i v e w i t h MS 2 0 4 e x i s t s i n A i d 2 0 4 . T h i s c o u l d b e a n e w s t r a t e g y t o o b t a i n c o n f o r m a t i o n a l e p i t o p e o f a g i v e n a n t i g e n a n d may s e r v e a s t h e a l t e r n a t i v e o f c o n t r a c e p t i v e v a c c i n e s . - i i i -T A B L E OF CONTENTS INTRODUCTION (page 1) E a r l y S t u d i e s (page 1) Sperm A n t i g e n - B a s e d C o n t r a c e p t i v e V a c c i n e R e s e a r c h (page 3) 1) . I n f e r t i l i t y and t h e Sperm A n t i b o d y (page 5) 2) . S t u d i e s on L D H - C 4 (page 6) 3) . Sperm Membrane A n t i g e n and Immunocontracept ion (page 9) 4) . A n t i - I d i o t y p i c A n t i b o d y - - An A l t e r n a t i v e (page 12) PART ONE. STUDIES OF SPERM ANTIGENS R E A C T I V E WITH THE MONOCLONAL ANTIBODIES THAT INHIBIT F E R T I L I T Y (page 16) M a t e r i a l s and Methods (page 16) Chemica ls (page 16) Animals (page 17) The M o n o c l o n a l A n t i b o d y a g a i n s t Mouse Sperm (page 17) P r o d u c t i o n of M o n o c l o n a l A n t i b o d y f r o m A s c i t e s F l u i d (page 18) I n d i r e c t i m m u n o f l u o r e s c e n t A s s a y and I n d i r e c t Immunof luorescent I n h i b i t i o n A s s a y (page 19) E n z y m e - l i n k e d immunosorbent a s s a y (page 20) I m m u n o - a f f i n i t y C h r o m a t o g r a p h y (page 21) Sodium D o d e c y l S u l f a t e - P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s (page 23) - iv -High P e r f r o m a n c e L i q u i d Chromatoghpphy (page 24) A c t i v e Immunization wi th the P u r i f i e d Sperm A n t i g e n (page 25) A n t i f e r t i l i t y S t u d i e s (page 26) S t a t i s t i c a l A n a l y s i s (page 28) R e s u l t s (page 29) A n t i g e n P u r i f i c a t i o n (page 29) A c t i v e Immunization with the P u r i f i e d A n t i g e n (page 31) In V i t r o F e r t i l i z a t i o n Experiment and .Sperm P e n e t r a t i o n A s s a y (page 32) D i s c u s s i o n (page 33) P u r i f i c a t i o n of the Sperm A n t i g e n (page 33) A c t i v e Immunization of the P u r i f i e d sperm a n t i g e n in Mouse (page 37) A n t i f e r t i l i t y E f f e c t of A c t i v e Immunization wi th the P u r i f i e d Sperm A n t i g e n (page 38) C o n c l u s i o n s (page 40) PART TWO. S t u d i e s of A n t i - i d i o t y p i c A n t i b o d i e s A g a i n s t a M o n o c l o n a l A n t i - s p e r m A n t i b o d y (page 4) M a t e r i a l s and methods (page 41) Chemica ls (page 41) Animals (page 41) M o n o c l o n a l A n t i b o d y a g a i n s t Mouse Sperm — MS 204 (page 41) Immunization wi th MS 204 in Rabbi t (page 42) v -P u r i f i c a t i o n of A n t i - I d i o t y p i c A n t i b o d y a g a i n s t MS 204 (page 42) Immunization wi th Aid 204 in Female Mice (page 44) E n z y m e - l i n k e d Immunosorbent A s s a y (page 44) R e s u l t s (page 46) G e n e r a t i o n of Rabbi t A n t i s e r u m a g a i n s t MS 204 (page 46) P u r i f i c a t i o n of Rabbi t A n t i s e r u m a g a i n s t t h e Fab Fragment of MS 204 (page 46) C h a r a c t e r i z a t i o n of Rabbit A id 204 (page 47) A c t i v e Immunization w i th A i d 204 in Mice (page 47) A n t i f e r t i l i t y E f f e c t of A n t i - A i d 204 a n t i s e r a (page 48) D i s c u s s i o n (page 49) C o n c l u s i o n s (page 52) APPENDIX. A SUUMARY OF GENERATION OF HYBRIDOMAS SECRETING MONOCLONAL ANTIBODIES OF IgA C L A S S AGAINST HUMAN SPERM (page 53) T a b b i e s (page 55) F i g u r e s (page 59) R e f e r e n c e s (page 68) L I S T OF T A B L E T a b l e I. P u r i f i c a t i o n of sperm a n t i g e n , M S A - 2 0 7 , f rom the mouse t e s t i s , (page 55) T a b l e II. I n h i b i t o r y e f f e c t of monoc lona l a n t i b o d y , MS 207, and the c o r r e s p o n d i n g isoimmune s e r a r a i s e d a g a i n s t the p u r i f i e d mouse sperm a n t i g e n , M S A - 2 0 7 , on in v i t r o f e r t i l i z a t i o n of mouse o v a . (page 56) T a b l e III. I n h i b i t o r y e f f e c t of isoimmune s e r a r a i s e d a g a i n s t p u r i f i e d sperm a c r o s o m a l a n t i g e n , M S A - 2 0 7 , on human sperm p e n e t r e a t i o n t o z o n a - f r e e h a m s t e r o v a . (page 57) T a l b e IV. I n h i b i t o r y e f f e c t of mouse a n t i - r a b b i t A id 204 and i t s c o r r e s p o n d i n g monoc lona l a n t i b o d y , MS 204, on in v i t r o f e r t i l i z a t i o n of mouse o v a . (page 58) - v i i -L I S T OF FIGURES F i g u r e 1. E l u t i o n p r o f i l e s f o r the p u r i f i c a t i o n of sperm a n t i g e n s r e a c t i v e t o MS 207 f rom mouse t e s t i s homogenate us ing an MS 207 immunoaf f in i ty column, (page 59) F i g u r e 2. The y i e l d of t o t a l a c t i v i t y of M S A - 2 0 7 d u r i n g t h e p r o c e s s of p u r i f i c a t i o n , (page 60) F i g u r e 3. The r e s u l t s of e n z y m e - l i n k e d immunosorbent a s s a y (ELISA) showing the s p e c i f i c r e a c t i o n of MS 207 t o the p u r i f i e d mouse sperm a n t i g e n , M S A - 2 0 7 . (page 61) F i g u r e 4. Sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s ( S D S - P A G E , 6% gel) showing the p u r i t y of M S A - 2 0 7 , a sperm a n t i g e n p u r i f i e d f r o m mouse t e s t i s homogenate by an MS 207 immunoaf f in i ty column, (page 62) F i g u r e 5. The e l u t i o n p r o f i l e f o r M S A - 2 0 7 from g e l f i l t r a t i o n - h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC). (page 6 3) - v i i i -F i g u r e 6. I n d i r e c t i m m u n o f l u o r e s c e n t a s s a y showing t h a t t h e isoimmune s e r a a g a i n s t M S A - 2 0 7 c r o s s r e a c t w i th human sperm (1,000X). (page 64) F i g u r e 7. Serum a n t i b o d y t i t e r s f o l l o w i n g immunizat ions wi th M S A - 2 0 7 in female mice, (page 65) F i g u r e 8. The r e s u l t s of e n z y m e - l i n k e d immunosorbent a s s a y (ELISA) showing the s p e c i f i c r e a c t i o n of A id 204 (ant ibody 2) t o t h e a n t i - A i d 204 (ant ibody 3). (page 66) F i g u r e 9. I n d i r e c t immunofluor e s c e n t a s s a y showing t h a t heteroimmune s e r a a g a i n s t A id 204 c r o s s r e a c t wi th mouse sperm, (page 67) - ix -LIST OF ABBREVIATIONS Aid 204: a n t i - i d i o t y p i c a n t i b o d y a g a i n s t Fab of MS 204 BSA: b o v i n e s e r u m albumin D E A E : d i e t h y l a m i n o e t h y l DMSO: D i m e t h y l s u l f o x i d e DOC: d e o x y c h o l a t e E D T A : e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d E L I S A : e n z y m e - l i n k e d immunosorbent a s s a y F a b : a n t i g e n b ind ing fragment of immunoglobuline F c : c o n s t a n t fragment of immunoglobuline F I T C : f l u o r e s c e i n e i s o t h i o c y a n a t e HAT: h y p o x a n t h i n e , a m i n o p t e r i n e and thymidine hCG: human c h o r i o n i c g o n a d o t r o p i n HPLC: high p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y HS 11: monoc lona l a n t i b o d y a g a i n s t human sperm No.11 HS 63: monoc lona l a n t i b o d y a g a i n s t human sperm No.63 HT: h y p o x a n t h i n e and thymidine IMDM: Dulbecco ' s modi f i ed Eag le ' s medium I.P.: i n t r a p e r i t o n e a l ( l y ) IVF: in v i t r o f e r t i l i z a t i o n LIZ: l i t h i u m d i i o d o s a l i c y l a t e - x -L P S : l i p o p o l y s a c c h a r i d e MS 204: monoc lona l a n t i b o d y a g a i n s t mouse sperm No. 204 MS 207: monoc lona l a n t i b o d y a g a i n s t mouse sperm No. 207 M S A - 2 0 7 - S (-P): mouse sperm a n t i g e n r e a c t i v e wi th MS 207, s o l u b l e or p a r t i c u l a t e form O.D.: o p o t i c a l d e n s i t y OPD: O - p h e n y l e n e diamine P A G E : p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s PBS: p h o s p h a t e b u f f e r e d s a l i n e P B S - B S A : PBS c o n t a i n i n g 0.5% BSA P E G : p o l y e t h y l e n e g l y c o l PMSG: p r e g n a n t mares ' s e r u m g o n a d o t r o p i n PMSF: p h e m y l n e t h y l s u l f o n y l f l u r o r i d e RPMI 1640: R u s e l l P a r k Memmorial I n s t i t u t e medium 1640 s . c : s u b c u t a n e o u s e ( l y ) SDS: sodium d o d e c y l s u l f a t e TEMED: N,N'-methylene b i s a c r y l a m i d e and NjNjN^N'- te tramethylemediamine UBC: the U n i v e r s i t y of B r i t i s h Columbia - x i -ACKNOWLEDGEMENT Thi s t h e s i s work has b e e n s u p e r v i s e d by D r . C h i - Y u G r e g o r y L e e . Without h is gu idance and f i n a n c i a l s u p p o r t , t h i s t h e s i s would not have r e a c h e d c o m p l e t i o n . His deep u n d e r s t a n d i n g and i n t e r e s t in t h e problems e n c o u n t e r e d t h r o u g h o u t the r e s e a r c h h a v e e n c o u r a g e d me t o e x p l o r e t h e i r n a t u r e wi th e x t e n s i v e e f f o r t s . I am a l s o g r a t e f u l t o D r s . D. Rurak , P. Hahn, J . L e v y and S. C. Sung f o r t h e i r d i s c u s s i o n s about the i d e a s and r e s u l t s of t h i s t h e s i s work. Dr . Y. Yang's he lp in t e c h n i q u e s at the i n i t i a l s t a g e of t h i s g r a d u a t e r e s e a r c h i s h igh ly a p p r e c i a t e d . F i n a l l y , I thank Mr. D. Chang and Mr. D. H s i a n g f o r t h e i r p r o o f r e a d i n g and g r a m m a t i c a l c o r e c t i o n s . - x i i -INTRODUCTION E a r l y S t u d i e s T h e r e has b e e n c o n s i s t e n t i n t e r e s t o v e r t h e y e a r s i n the p o s s i b l e i n d u c t i o n of i n f e r t i l i t y by immunizat ion a g a i n s t sperm. As e a r l y as t h e beg inn ing of the c e n t u r y , i t has b e e n d e m o n s t r a t e d t h a t s p e r m c e l l s a r e a n t i g e n i c and c a p a b l e of e l i c i t i n g a u t o - and i so immuni ty [see r e v i e w by Menge, 1980]. It i s known t h a t the immune s y s t e m is f u l l y d e v e l o p e d p r i o r t o s p e r m a t o g e n e s i s i n p u b e r t y [Tung, 1980; Tung et a l , 1980; H e r r et a l , 1985]. Due t o the e x i s t a n c e of the b l o o d - t e s t i s b a r r i e r , s p e r m a t o g e n e s i s i s e n t i r e l y i s o l a t e d f r o m the immune s y s t e m p r e s e n t in t h e c i r c u l a t i o n [ P a r v i n e n , 1982]. As a c o n s e q u e n c e , s p e r m a n t i g e n s a r e p e r c e i v e d as "fore ign" t o our body's immune s y s t e m . E a r l y s t u d i e s in e x p e r i m e n t a l animals s u g g e s t e d i n f e r t i l i t y c o u l d be i n d u c e d by immunizat ion of f emales wi th semen [see r e v i e w by K a t s h , 1959] . T h i s e a r l y r e p o r t s t i m u l a t e d a s e r i e s of a t t e m p t s t o induce an immune r e s p o n s e t o human s p e r m a n t i g e n s in women [ R o s e n f i e l d , 1926; Basken , 1932], which c o u l d r e s u l t i n i n f e r t i l i t y f o l l o w i n g immunizat ions wi th semen. T h e s e s t u d i e s d e m o n s t r a t e d the i s o — i m m u n o g e n i c i t y of human sperm in women and i n t r o d u c e d the c o n c e p t of an immunologic c o n t r a c e p t i v e b a s e d on sperm a n t i g e n s . - 1 -However , t h e s e o b s e r v a t i o n s were not s u b s t e n t i a t e d u n t i l the 1950's and 1960's when s e v e r a l i n v e s t i g a t o r s r e p o r t e d the p o s s i b l e a s s o c i a t i o n b e t w e e n t h e p r e s e n c e of sperm a n t i b o d i e s and human i n f e r t i l i t y [Rumke and H e l l e u g a , 195 F j a l l b r a n t , 1968; F r a n k l i n and Dukes , 1964] . A s i g n i f i c a n t n e g a t i v e a s s o c i a t i o n b e t w e e n s e r u m t i t e r s of a n t i s p e r m a n t i b o d i e s and c a p a c i t y of f e r t i l i t y was r e p o r t e d in an e p i d e m e l o g i c a l s u r v e y [see r e v i e w by Menge, 1980]. S i m i l a r l y , the p r e s e n c e of l o c a l l y s e c r e t e d sperm a n t i b o d i e s h a v e b e e n found in t h e c e r v i c a l mucus of i n f e r t i l e women [Menge et a l , 1982; P a s l o w et a l , 1985] . It was a l s o r e p o r t e d t h a t c o r t i c o s t e r o i d t h e r a p y , which r e d u c e s a n t i b o d y t i t e r s , has r e s u l t e d in s u c c e s s f u l p r e g n a n c y i n 30 t o 40% of t h e t r e a t e d c o u p l e s [ H a r g r e a v e and E l t o n , 1982; Shulman and Shulman, 1981]. T h e s e c l i n i c a l o b s e r v a t i o n s p r o v i d e d a s t r o n g b a s i s f o r t h e deve lopment of sperm a n t i g e n b a s e d i m m u n o c o n t r a c e p t i v e v a c c i n e s . E x p e r i m e n t a l animal s t u d i e s h a v e shown c o n t r a c e p t i v e p o t e n t i a l by means of immunizat ion wi th s p e r m - r e l a t e d a n t i g e n s . One example i s e x p e r i m e n t a l a l l e r g i c o r c h i t i s (EAO), a w e l l - s t u d i e d model c o n c e r n i n g the i n d u c t i o n of autoimmune d i s e a s e by immunizat ion wi th a u t o l o g o u s or h e t e r o l o g o u s s p e r m or t e s t i s e x t r a c t [Freund et a l 1953] . In t h i s model, i t was s u g g e s t e d t h a t b o t h humora l and c e l l u a r immunity were i n v o l v e d . T h i s may be due t o the immune a t t a c k - 2 -through the rete testis and deferas tubules which are considered weak points along the blood-testis barrier [Jones, 1981]. However, only some of the sperm surface antigens can induce EAO. Voisin et al [1974] have isolated and characterized three preparations of aspermatogenic antigen from guinea pigs. One of them is localized in the plasma membrane and can induce the secretion of sperm immobilizing antibodies in experimental animals. In the female experimental animals various degrees of immunity and infertility could be induced by injections with sperm or testis antigens [Bell, 1969; Menge 1970]. As reviewed by Jones [1982], numerous mechanisms are involved in immunologic infertility in experimental female animals. These include immobilization and death of sperm, agglutination, interference with sperm penetration of mucus and sperm transport, increased phagocytosis, and interference with sperm-ovum contact and interaction. In summary, early studies have proven that 1), naturally occurring sperm antibodies correlate with human infertility; 2), some sperm components are auto- and/or isoimmunogenic, and 3), immunization with sperm or testis preparations can induce infertility. These studies established the basis for research on immunologic contraception. Sperm Antigen-Based Contraceptive Vaccine Research - 3 -P r o g r e s s i n a n t i - s p e r m v a c c i n e deve lopment has b e e n s low d e s p i t e the e x t e n s i v e animal s t u d i e s . One r e a s o n c o u l d be due t o the f a c t t h a t t h e a n t i g e n s d e f i n e d in e x p e r i m e n t a l animals a r e u s u a l l y s p e c i e s - s p e c i f i c and t h e i r human c o u n t e r - p a r t s a r e d i f f i c u l t t o i s o l a t e and c h a r a c t e r i z e . However , d e s p i t e t h e s e d i f f i c u l t i e s the b a s i c p r i n c i p l e of immunologic c o n t r a c e p t i o n remains a t t r a c t i v e [Alexander and A n d e r s o n , 1983]. In c o n t r a s t wi th o t h e r t e c h n i q u e s , immunologic c o n t r a c e p t i v e methods o f f e r t h e f o l l o w i n g unique a d v a n t a g e s : t h e y a r e a p h y s i o l o g i c a l methods in t h e s e n s e t h a t t h e y do not r e q u i r e c o n s t a n t m e d i c a t i o n wi th s y n t h e t i c compounds and t h e y r e q u i r e only p e r i o d i c i n t a k e . F u r t h e r m o r e , the amount of a n t i g e n a d m i n i s t e r e d i s r e l a t i v e l y smal l compared wi th o t h e r chemica l and hormonal methods f o r c o n t r a c e p t i o n . B a s i c a l l y , t h e r e a r e f o u r a p p r o a c h e s t o o b t a i n i n g sperm a n t i g e n s f o r v a c c i n e deve lopment . One i s t o i d e n t i f y t h o s e sperm a n t i g e n s t h a t e l i c i t n a t u r a l l y o c c u r r i n g sperm a u t o - or i s o a n t i b o d i e s in human i n f e r t i l e c o u p l e s . S t u d i e s and e x p e r i e n c e h a v e shown t h a t t h e a u t o - o r isoimmune r e s p o n s e s t o human sperm a r e v e r y h e t e r o g e n e o u s [Jones , 1982], thus i t i s r e l a t i v e l y d i f f i c u l t t o i d e n t i f y sperm a n t i g e n s f r o m t h i s a p p r o a c h . The s e c o n d a p p r o a c h is t o u s e wel l c h a r a c t e r i z e d s p e r m - s p e c i f i c enzymes f o r v a c c i n e deve lopment . A l t h o u g h most enzymes a r e not l o c a l i z e d on the s p e r m s u r f a c e , some of them a r e found on the s u r f a c e and t h e y c a n induce immune r e s p o n s e s t h a t a f f e c t normal sperm f u n c t i o n s . One s u c h enzyme is l a c t a t e d e h y d r o g e n a s e - C 4 ( L D H -C4) , a we l l c h a r a c t e r i z e d , s p e r m - s p e c i f i c g l y c o l y t i c enzyme t h a t has b e e n s t u d i e d as a p o t e n t i a l immunogen f o r s p e r m a n t i g e n - b a s e d v a c c i n e . Th i s w i l l be ment ioned l a t e r i n t h i s c h a p t e r . The t h i r d a p p r o a c h is t o g e n e r a t e monoc lona l a n t i b o d i e s a g a i n s t sperm s u r f a c e a n t i g e n s . By u s i n g g e n e r a t e d sperm monoc lona l a n t i b o d i e s , sperm s u r f a c e a n t i g e n s can be i d e n t i f i e d , i s o l a t e d , p u r i f i e d and c h a r a c t e r i z e d . T h e i r f u n c t i o n a l r o l e s d u r i n g f e r t i l i z a t i o n p r o c e s s e s can be a s s e s s e d . The sperm a n t i g e n r e a c t i v e t o t h e s e monoc lona l a n t i b o d i e s c o u l d be s e l e c t e d f o r v a c c i n e deve lopment . The f o u t h a p p r o a c h would be t o g e n e r a t e a n t i - i d i o t y p i c a n t i b o d y t o sperm a n t i b o d i e s . A c c o r d i n g t o r e c e n t s t u d i e s , a n t i - i d i o t y p i c a n t i b o d i e s sometimes c a n e x p r e s s t h e " i n t e r n a l image" of t h e immunogen [ J e r n e , 1 9 7 4 ] , t h e r e f o r e the a n t i - i d i o t y p i c a n t i b o d y c o u l d s e r v e as an a n t i g e n and induce a n t i s p e r m a n t i b o d i e s . In t h i s c h a p t e r , r e c e n t s t u d i e s in the f o u r a p p r o a c h e s w i l l be r e v i e w e d , and t h e t h i r d a p p r o a c h , which i s d i r e c t l y r e l a t e d t o t h e o b j e c t i v e s of t h e s i s , w i l l be r e v i e w e d in d e t a i l . 1. I n f e r t i l i t y and t h e Sperm A n t i b o d y S t u d i e s of a n t i b o d y r e s p o n s e s in i n f e r t i l e humans have - 5 -been e x t e n s i v e l y r e v i e w e d by Menge [1980a] and M e t h l e r [1980]. B i o a s s a y s i n v o l v i n g the a g g l u t i n a t i o n and complement dependent Immobi l izat ion of sperm have b e e n u s e d in b l o o d and g e n i t a l t r a c t s e c r e t i o n s t o d e m o n s t r a t e the p r e s e n c e of sperm a n t i b o d i e s . T h e s e s t u d i e s have been f u r t h e r c o n f i r m e d by a n t i b o d y l o c a l i z a t i o n t e c h n i q u e s , s u c h as the i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y . By u s i n g t h e s e methods , s p e r m a n t i b o d i e s d e t e c t e d in t h o s e i n f e r t i l e p a t i e n t s were found t o b ind wi th v a r i o u s p a r t s of sperm s u r f a c e . T h e s e i n c l u d e the a c r o s o m e , e q u a t o r i a l segment , p o s t n u c l e a r cap , m i d - p i e c e , main t a l l - p i e c e and t a i l e n d - p i e c e [Dondero , 1986; L e n z i , 1986]. S u r f a c e a n t i g e n s of t h e a c r o s o m e and main t a i l - p i e c e a p p e a r t o p r o v o k e sperm a n t i b o d i e s of s p e c i a l r e l e v a n c e t o human i n f e r t i l i t y [Jones , 1982], but p r e v i o u s s t u d i e s a l s o showed t h a t some of t h e s e sperm a n t i b o d i e s c r o s s - r e a c t wi th a n t i g e n s f rom o t h e r t i s s u e s [Tung, 1976; Tung et a l , 1976]. F o r example, some a u t o - a n t i b o d i e s a g a i n s t a c t i n in smooth musc le a l s o c r o s s r e a c t w i th t h e p o s t a c r o s o m a l r e g i o n of the sperm [ C l a r k e et a l 1977]. A n t i - s m o o t h musc le a n t i b o d i e s a r e r e l a t i v e l y common in a d u l t s , and a r e p r e s e n t i n h igh i n c i d e n c e in i n f e r t i l e women [Jones et a l 1981]. Due t o the i n t r i n s i c h e t e r o g e n e i t y of a u t o - or isoimmune r e s p o n s e t o sperm a n t i g e n s , i t i s d i f f i c u l t t o i d e n t i f y unique a n t i g e n s f o r v a c c i n e deve lopment by t h i s s t r a t e g y [Jones , 1982]. 2. S t u d i e s on L D H - C 4 - 6 -Sperm s p e c i f i c enzymes a r e p o t e n t i a l t a r g e t s f o r s p e r m a n t i g e n b a s e d v a c c i n e . Sperm a c r o s i n and h y a l u r o n i d a s e showed e a r l y p r o m i s e , but i t was d e m o n s t r a t e d l a t e r t o be u n s u i t a b l e [ A n d e r s o n and A l e x a n d e r , 1983; J o n e s , 1986]. Immune r e s p o n s e s can be Induced t o w a r d s t h e s e a n t i g e n s in mice, r a b b i t s and sheep , but t h i s has not b e e n a s s o c i a t e d wi th s i g n i f i c a n t a n t i f e r t i l i t y e f f e c t s . On t h e o t h e r hand, L D H - C 4 , a l s o known as L D H - X , a p p e a r s t o be a unique s p e r m - s p e c i f i c a n t i g e n . S u b s t e n t i a l p r o g r e s s has b e e n made t o w a r d s i t s a p p l i c a t i o n as an a n t i g e n in a c o n t r a c e p t i v e v a c c i n e [Goldberg , 1971; 1977; G o l d b e r g et a l , 1981]. L D H - C 4 i s d i s t i n c t f rom o t h e r L D H i soezymes found in v a r i o u s t i s s u e s and i t s chemica l and b i o l o g i c a l p r o p e r t i e s h a v e b e e n s t u d i e d e x t e n s i v e l y [L iang et a l , 1986] . T h i s enzyme f i r s t a p p e a r s in the p r i m a r y s p e r m a t o c y t e d u r i n g s p e r m a t o g e n e s i s . In m a t u r e sperm i t i s l o c a l i z e d i n s i d e t h e m i d - p i e c e and a l s o t h e s u r f a c e . It has many c h a r a c t e r i s t i c enzyme p r o p e r t i e s and c o n s t i t u t e s 90% of the L D H a c t i v i t y in sperm. By r e g u l a t i n g p y r u v a t e l e v e l s , i t may c o n t r i b u t e t o t h e c o n t r o l of sperm r e s p i r a t i o n and m o t i l i t y . L D H - C 4 has b e e n p u r i f i e d f rom the t e s t i s in mouse, r a t , r a b b i t , b u l l and man, and f rom human sperm. The comple te s e q u e n c e of murine L D H - C 4 has now b e e n d e t e r m i n e d and knowledge i s a v a i l a b l e c o n c e r n i n g i t s s e c o n d a r y and t e r t i a r y s t r u c t u r e [Musick and Rossmann, 1979; L i et a l , 1983]. The DNA s e q u e n c e f o r cod ing t h e molecu le of L D H - C 4 has a l s o b e e n d e t e r m i n e d r e c e n t l y [Wu - 7 -et al, 1987]. LDH-C4 is both aut©immunogenic and isoimmunogenic in experimental animals. Active and passive immunization of mice results in encouraging, though incomplete ahtifertility effects, which appear to operate at several stages in the reproductive processes: prefertilization, postfertilization and postimplantatlon [Jones, 1986]. The major effect, however, appears to be at the prefertilization stage. Although LDH-C4 is a cytoplasmic enzyme, the antigen is also found on the sperm surface. This may be due to secondary adsorption of LDH-C4 onto intact sperm released from damaged sperm, and diffusing from the sperm mid-piece onto tail membrane [Baylor et al, 1985]. Sperm can be agglutinated by anti-LDH-C4 antibody in vitro. Incubation of sperm with LDH-C4 antibody and complement causes disruption of the sperm membrane [Lerum and Goldberg, 1974; Goldberg et al, 1981]. Studies in baboons, immunized against LDH-C4 showed that 9 immunized baboons remained infertile in 22 of 30 matlngs (73%) [Goldberg et al, 1981], and there were no instances of recognisable abortion and the animals remained normal in ovulatory cycles. The antifertility effect appear to be positively correlated with antibody titers, and is reversible. In one of their recent reports Goldberg and his co-workers [Goldberg and Shelton, 1986] have mapped 8 antibody binding peptides from LDH-C4 subunits by trypsin - 8 -d i g e s t i o n and RIA. T h r e e of the p e p t i d e s were c h e m i c a l l y s y n t h e s i z e d . Immunization wi th t h e s e p e p t i d e s c o n j u g a t e d wi th c a r r i e r p r o t e i n was p e r f o r m e d t o r a i s e a n t i s e r a in female r a b b i t s . The a n t i s e r a were found t o c r o s s r e a c t wi th n a t i v e L D H - C 4 . Th i s s t u d y s u g g e s t a s t r a t e g y t o u t i l i z e a s y n t h e t i c a n t i g e n f o r c o n t r a c e p t i v e v a c c i n e deve lopment . 3. Sperm Membrane A n t i g e n and Immunocontracept ion Sperm membrane components p r o v i d e the b e s t t a r g e t f o r i m m u n o c o n t r a c e p t i o n . In r e c e n t y e a r s e f f o r t s have been made t o i s o l a t e a n t i g e n s i n s o l u b i l i z e d sperm membrane e x t r a c t s . It i s now a p p a r e n t from s t u d i e s u s i n g monoc lona l a n t i b o d i e s t h a t at l e a s t some sperm a n t i g e n s h a v e e p i t o p e s t h a t h a v e been c o n s e r v e d among s p e c i e s . Th i s has widened the s c o p e f o r p r o g r e s s in t h i s f i e l d whereby sperm a n t i g e n s f rom human t i s s u e c a n be s e l e c t e d and s t u d i e d in l a b o r a t o r y a n i m a l s . C o n v e r s e l y , sperm a n t i g e n s i d e n t i f i e d f r o m l a b o r a t o r y animals may c o n t a i n e p i t o p e s in common with a human sperm membrane component . T h e r e f o r e t h i s method c a n be u s e d f o r t h e i s o l a t i o n and c h a r a c t e r i z a t i o n of a n t i g e n i c d e t e r m i n a n t s f o r c o n t r a c e p t i v e v a c c i n e deve lopment in human. Rabbi t sperm a u t o - a n t i g e n s (RSA1, 2 and 3) have b e e n s t u d i e d by O'Rand et a l [1984a, 1984b]. They a r e low m o l e c u l a r weight (10 -13 KDa) g l y c o p r o t e i n s i s o l a t e d f r o m the p lasma membrane of r a b b i t sperm. A n t i b o d i e s r a i s e d a g a i n s t - 9 -RSA i n h i b i t f e r t i l i z a t i o n p r o b a b l y by i n t e r f e r i n g wi th sperm p e n e t r a t i o n ; immediate ly a f t e r f e r t i l i z a t i o n the RSA complex c a n be d e m o n s t r a t e d on t h e p lasma membrane of the egg. M o n o c l o n a l a n t i - s p e r m a n t i b o d i e s c a n be a p o w e r f u l t o o l t o i d e n t i f y , p u r i f y and c h a r a c t e r i z e sperm a n t i g e n s . The a d v a n t a g e of t h i s a p p r o a c h is t h a t unique sperm a n t i g e n s can be s e l e c t e d t h r o u g h the f u n c t i o n a l a s s a y of monoc lona l sperm a n t i b o d i e s . Dur ing r e c e n t y e a r s , a n t i - s p e r m monoc lona l a n t i b o d i e s have b e e n u s e d i n d e v e l o p m e n t a l s t u d i e s [Lee et a l , 1986a] , and t o d e t e c t s u r f a c e marker changes of s p e r m d u r i n g c a p a c i t a t i o n / a c r o s o m e r e a c t i o n p r o c e s s e s [Menge et a l , 1983; Wolf et a l , 1985; Myles et a l ; 1984]. A l t h o u g h s t u d i e s in t h i s a r e a a r e s t i l l a t an e a r l y s t a g e , s e v e r a l i n v e s t i g a t o r s have r e p o r t e d e n c o u r a g i n g r e s u l t s . U s i n g a monoc lona l a n t i b o d y as an immunoaf f in i ty l i g a n d , Naz et a l have i s o l a t e d a 63 K g l y c o p r o t e i n (GA-1) f r o m r a b b i t t e s t i s [1984] and a 23 K g l y c o p r o t e i n ( F A - 1 ) f rom s o l u b i l i z e d human t e s t e s [1986]. F A - 1 i s l o c a l i z e d on the p o s t a c r o s o m e , mid p i e c e and t a i l of t h e sperm in human, mouse, r a b b i t , b u l l and monkey. As r e p o r t e d , a n t i b o d i e s t o F A - 1 i n h i b i t f e r t i l i z a t i o n by b l o c k i n g i n t e r a c t i o n s b e t w e e n the sperm and t h e zona p e l l u c i d a . Up t o now, t h e n o t a b l e a n t i g e n s i s o l a t e d f rom s p e r m a r e RSA from r a b b i t [O'Rand et a l , 1984], G A - 1 f r o m r a b b i t [Naz - 10 -et a l , 1984], F A - 1 f rom human [Naz et a l , 1986] and L D H - C 4 [Go ldberg et a l , 1981] . But in l a b o r a t o r y animals none of the a n t i g e n s were ab le t o e l i c i t a 90-100% a n t i f e r t i l i t y e f f e c t . S ince 1981 L e e and h i s a s s o c i a t e s have g e n e r a t e d numerous monoc lona l a n t i b o d i e s t o human, mouse and r a b b i t sperm [1982a,b; 1983; 1984a,b,c; 1985] . The a n t i f e r t i l i t y e f f e c t s of t h e s e monoc lona l a n t i b o d i e s have been a s s e s s e d . The immunoglobulin s u b c l a s s e s of t h e a n t i b o d i e s and c y t o l o g i c a l l o c a t i o n of r e l e v a n t sperm a n t i g e n s and t i s s u e s p e c i f i c i t y have b e e n s t u d i e d [Lee et a l , 1983] . They h a v e a l s o o b s e r v e d the c h a r a c t e r i z a t i o n of some of the r e l e v a n t sperm a n t i g e n s by s e v e r a l c h e m i c a l and enzymat i c t r e a t m e n t s f o l l o w e d by t h e i n d i r e c t immunofluor e s c e n t a s s a y [Lee et a l , 1983; 1984b]. F r o m among t h e s e a n t i b o d i e s , a few c a n d i d a t e s were s e l e c t e d f o r f u n c t i o n a l and d e v e l o p m e n t a l s t u d i e s . In p a r t i c u l a r , t h e f o l l o w i n g c h a r a c t e r i s t i c s were s t u d i e d : t h e i n h i b i t o r y e f f e c t s on f e r t i l i z a t i o n in v i t r o and in v i v o [Lee et a l , 1986b], t h e a p p e a r a n c e of t h e c o r r e s p o n d i n g a n t i g e n s d u r i n g s p e r m a t o g e n e s i s and s p e r m m a t u r a t i o n [Lee et a l , 1986a] , and the c o m p a r i s o n of t h e i r a n t i f e r t i l i t y e f f e c t a d m i n i s t e r e d in male and female mice [Lee et a l , 1987]. T h e s e o b s e r v a t i o n s p r o v i d e d a b a s i s f o r c a r r y i n g out t h i s p r o j e c t f o r p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of s p e r m a n t i g e n s by means of monoc lona l a n t i b o d i e s as p r o b e s . In f a c t , t h i s p r o j e c t i s a p a r t of the l o n g t e r m e f f o r t of the - 11 -L a b o r a t o r y of A n d r o l o g y at UBC t o d e v e l o p a workable s p e r m a n t i g e n - b a s e d c o n t r a c e p t i v e v a c c i n e f o r u s e in fami ly p l a n n i n g . 4. A n t i - i d i o t y p i c A n t i b o d y - - an A l t e r n a t i v e In the c a s e t h a t the a n t i g e n i c d e t e r m i n a n t r e a c t i n g wi th t h e monoc lona l a n t i b o d y is c o n f o r m a t i o n a l or c a r b o h y d r a t e i n n a t u r e , i t might be d i f f i c u l t t o m a s s - p r o d u c e t h e i r a n t i g e n i c f ragments by recombinent DNA t e c h n o l o g y in the f u t u r e . An a l t e r n a t i v e a p p r o a c h t o o b t a i n i n g a n t i g e n f o r i m m u n o c o n t r a c e p t i o n would be t h e g e n e r a t i o n of a n t i - i d i o t y p i c a n t i b o d i e s . A c t u a l l y the "antigen" o b t a i n e d by t h i s means would be the "image" of the o r i g i n a l a n t i g e n . It has b e e n documented t h a t some a n t i - i d i o t y p i c a n t i b o d i e s e x p r e s s t h e " i n t e r n a l image" of a n t i g e n s t o which the h o s t has b e e n i n i t i a l l y immunized [ J e r n e , 1974;]. B r i e f l y , an i d i o t y p e i s a s e t of d e t e r m i n a n t s ( i d i o t o p e s ) e x p r e s s e d in the v a r i a b l e domain, or , g e n e r a l l y r e g a r d e d as t h e a n t i g e n b ind ing s i t e of an a n t i b o d y [ F a r i s and L o , 1985]. An i d i o t y p e c a n i t s e l f a c t as an a n t i g e n f rom which a n o t h e r a n t i b o d y ( a n t i - i d i o t y p e ) may a r i s e . The r e l a t i o n b e t w e e n i d i o t y p e s and a n t i - l d i o t y p e s a r i s i n g in t h e c o u r s e of immune r e s p o n s e has been f i r m l y e s t a b l i s h e d [Rawley et a l , 1980]. Assuming t h a t an a n t i - i d i o t y p e c o m p r i s e s an i n t e r n a l image of a g i v e n a n t i g e n i c domain, t h e a n t i - i d i o t y p e t o a l i g a n d (e.g. - 12 -hormone) s h o u l d exhib i t b i o l o g i c a l a n d / o r r e c e p t o r b inding a c t i v i t y a t t h e c o r r e s p o n d i n g r e c e p t o r . T h e o r e t i c a l l y , only when the immunogenic e p i t o p e of a g i v e n l i g a n d c o i n c i d e s wi th the b ind ing s i t e of the hormone, w i l l an a n t i - i d i o t y p e h a v e t h e a n t i - r e c e p t o r a c t i v i t y . So f a r , the a n t i - i d i o t y p i c a n t i b o d i e s wi th a n t i - r e c e p t o r a c t i v i t y c o v e r a wide range of l i g a n d s , i n c l u d i n g i n s u l i n [Sege and P e r t e r s o n , 197 8], T S H [Islam et a l , 1983] and an a d r e n e r g i c a n t a g o n i s t [ S e h r e i b e r et a l , 1980]. The s e c o n d p a r t of t h i s t h e s i s work i s d e d i c a t e d t o g e n e r a t e a n t i - i d i o t y p i c a n t i b o d y t o a s e l e c t e d sperm monoc lona l a n t i b o d y . The s p e c i f i c aims of t h i s p r o j e c t i s t o o b t a i n the "image" of a s p e c i f i c sperm a n t i g e n t h r o u g h the g e n e r a t i o n of a n t i - i d i o t y p i c a n t i b o d i e s . A t t e m p t s were made t o s e e i f the a n t i - i d i o t y p i c a n t i b o d i e s which e x p r e s s i n t e r n a l images of sperm c a n s e r v e as an a l t e r n a t i v e f o r c o n t r a c e p t i v e v a c c i n e . In summary, f o u r a p p r o a c h e s t o o b t a i n i n g sperm a n t i g e n s f o r i m m u n o c o n t r a c e p t i o n have b e e n r e v i e w e d . The o b j e c t i v e of t h i s t h e s i s i s t o o b t a i n sperm a n t i g e n by means of immunoaf f in i ty c h r o m a t o g r a p h y u s i n g monoc lona l sperm a n t i b o d i e s as immunoligand, and t o g e n e r a t e a n t i - i d i o t y p i c a n t i b o d y t o s p e r m a n t i b o d y . A c t i v e immunizat ion wi th p u r i f i e d sperm a n t i g e n s and wi th o b t a i n e d sperm a n t l - i d i o t y p e s w i l l be c a r r i e d out , and the a n t i f e r t i l i t y e f f e c t of t h e immunizat ions w i l l be a s s e s s e d . In the appendix of t h i s t h e s i s , some p r e l i m i n a r y e f f o r t s in g e n e r a t i n g a n t i - s p e r m I g A - s e c r e t i n g hybr idomas wi l l be r e p o r t e d . In males , due t h e e x i s t a n c e of b l o o d - t e s t i s and b l o o d - e p l d i d y m i s b a r r i e r s , the g e n i t a l t r a c t i s i s o l a t e d f r o m c i r c u l a t i n g a n t i b o d i e s [Ewing and Chang, 1986]. Recent s t u d i e s have i n d i c a t e d t h a t s e c r e t o r y or l o c a l a n t i b o d i e s (IgA or IgG ) from the p r o s t a t e may p l a y Important r o l e s in human I n f e r t i l i t y [ C l a r k e et a l , 1985;]. In the female , l o c a l l y s e c r e t e d IgA f r o m the c e r v i x was shown t o r e d u c e the a b i l i t y of sperm t o p e n e t r a t e c e r v i c a l mucus [ C l a r k e , 1984; C l a r k e et a l , 1984]. Menge and his c o - w o r k e r s [Naz et a l , 1985] found t h a t in female r a b b i t s more a n t i b o d i e s of IgA c l a s s were i n d u c e d l o c a l l y in t h e r e p r o d u c t i v e t r a c t a g a i n s t sperm a n t i g e n s r a t h e r t h a n t h o s e of IgG c l a s s . B a s e d on t h e s e o b s e r v a t i o n s i t seems wor thwhi l e t o g e n e r a t e hybr idomas s e c r e t i n g monoc lona l a n t i b o d i e s of the IgA c l a s s . One c o u l d c a r r y out a d e t a i l e d s t u d y on the mechanism of the a c t i o n of IgA on sperm t o f u r t h e r u n d e r s t a n d the immunological f a c t o r s a s s o c i a t e d w i th human i n f e r t i l i t y . A c c o r d i n g t o p r e v i o u s r e p o r t s [ E l s o n and E a l d i n g 1984a,b; Taubman et a l 1983], IgA s e c r e t i n g hybr idomas c o u l d - 14 -be e s t a b l i s h e d by u s i n g l y m p h o c y t e s f rom P e y e r ' s p a t c h e s and m e s e n t e r y lymph nodes of B A L B / c mice t h r o u g h o r a l immunizat ion wi th a p p r o p r i a t e a d j u v a n t s . In p r i n c i p l e , a n t i g e n s ga in a c c e s s t o g u t - a s s o c i a t e d lymphoid t i s s u e t h r o u g h s p e c i a l i z e d m i c r o f o l d e d e p i t h e l i a l c e l l s o v e r l y i n g P e y e r ' s p a t c h e s . F o l l o w i n g T c e l l p r o c e s s i n g and T c e l l - B c e l l c o l l a b o r a t i o n i n the P e y e r ' s p a t c h e s , t h e s t i m u l a t e d B c e l l s undergo b l a s t t r a n s f o r m a t i o n and s u b s e q u e n t l y c i r c u l a t e in the s y s t e m i c c i r c u l a t i o n and t h e n home t o v a r i o u s m u c o s a l t i s s u e s , i n c l u d i n g g u t - a s s o c i a t e d lymph nodes and g e n i t o - u r i n a r y t r a c t . T h e r e f o r e , l y m p h o c y t e s r e c o v e r e d f r o m g u t - a s s o c i a t e d lymph nodes f o l l o w i n g o r a l immunizat ion s h o u l d be t h e e a s i e s t way t o g e n e r a t e I g A - s e c r e t i n g h y b r i d o m a s . - 15 -PART ONE. STUDIES OF SPERM ANTIGENS R E A C T I V E WITH MONOCLONAL ANTIBODIES THAT INHIBIT F E R T I L I T Y MATERIALS AND METHODS Chemica l s D i m e t h y l s u l f o x i d e (DMSO), l i p o p o l y s a c c h a r i d e (LPS), m e t h y l c e l l u l o s e , b o v i n e s e r u m albumin (BSA), l ac moid , c h l o r a m i n e T, sodium d i s u l f i t e , nonident P - 4 0 (NP-40) , c o l l a g e n a s e , h y a l u r o n i d a s e , t r y p s i n , human c h o r i o n i c g o n a d o t r o p i n (hCG), p r e g n a n t mares ' s e r u m g o n a d o t r o p i n (PMSG), a l p h a - t h i o g l y c e r o l , t r i s b a s e , sodium d e o x y c h o l a t e , c y a n o g e n bromide , comple te and incomple te F r e u n d ' s a d j u v a n t were p u r c h a s e d f r o m Sigma Chemica l Company, S t . L o u i s , Mo.. C e l l c u l t u r e media i n c l u d i n g IMDM (Dulbecco's modi f i ed Eag le ' s medium), RPMI 1640, p e n i c i l l i n - s t r e p t o m y c i n 100X and g lutamine were f r o m GIBCO, B u r l i n g t o n , C a n a d a . T i s s u e c u l t u r e p l a t e s and s e l e c t i o n media, i n c l u d i n g HAT (hypoxanth ine , a m i n o p t e r i n e and thymid ine , 100X s t o c k ) and HT (hypoxanth ine and thymidine , 50X s t o c k ) were from Flow L a b o r a t o r i e s , M i s s i s s a u g a , O n t a r i o , C a n a d a . F l u o r e s c e i n e i s o t h i o c y a n a t e ( F I T C ) - l a b e l e d goat a n t i - m o u s e IgG+M+A was f r o m C a p p e l / W a s h i n g t o n , M a l v e r n , PA. . A l l of t h e a n a l y t i c a l g r a d e r e a g e n t s r e q u i r e d f o r a c r y l a m i d e ge l e l e c t r o p h o r e s i s were from B i o - R a d L a b o r a t o r y , Richmond, CA. . N i t r o c e l l u l o s e f i l t e r p a p e r (0.45 um p o r e s i z e ) was f rom M i n i p o r e C o r p . , H a y w a r d , CA. I o d i n e - 1 2 5 (50 mCi/mmole) was f rom Amer sham, UK. P o l y e t h y l e n e g l y c o l (PEG) 1500 and sodium d o d e c y l s u l f a t e (SDS) were from B r i t i s h Drug House Chemica l L t d . , P o o l e , E n g l a n d . N,N'-methylene b i s a c r y l a m i d e and N ,N ,N' ,N ' - t e t ramethy lened iamine (TEMED) were p u r c h a s e d f rom E a s t m a n - K o d a k , N.Y. , USA. S e p h a r o s e 4B was f r o m P h a r m a c i a , U p p s a l a , Sweden. Animals Female mice of B A L B / c s t r a i n (6 - 10 weeks old) were p u r c h a s e d from J a c k s o n L a b o r a t o r y , Bar H a r b o r , Me.. Randomly b r e d C D - I mice (about 12 weeks old) and go lden h a m s t e r s of L V G s t r a i n were from C h a r l e s R i v e r Inc, Quebec , C a n a d a . M o n o c l o n a l A n t i b o d i e s a g a i n s t Mouse and Human Sperm The hybr idoma c e l l l i n e s e c r e t i n g monoc lona l a n t i b o d y a g a i n s t mouse sperm, MS 207, was s e l e c t e d f r o m a p o p u l a t i o n of hybr idomas g e n e r a t e d by L e e and h i s a s s o c i a t e s [Lee , et a l , 1986b]. MS 207 was shown t o r e a c t w i th mouse sperm a c r o s o m a l a n t i g e n , w i thout c r o s s - r e a c t i v i t y t o o t h e r t i s s u e s in the mouse or sperm from any o t h e r s p e c i e s . Th i s a n t i b o d y e x h i b i t s a s t r o n g i n h i b i t o r y e f f e c t on mouse f e r t i l i z a t i o n in v i t r o and ln v i v o [Lee et a l , 1986b]. P r o d u c t i o n of monoc lona l a n t i b o d i e s f r o m a s c i t e s f l u i d Mass p r o d u c t i o n of t h e s e l e c t e d monoc lona l a n t i b o d i e s was a c h i e v e d by i n d u c i n g a s c i t e s f l u i d s f rom B A L B / c mice a c c o r d i n g t o the f o l l o w i n g methods [Lee et a l , 1982a]: P r i s t a n e 0.5 ml was i n j e c t e d I.P. a t l e a s t 2 d a y s in a d v a n c e t o i r r i t a t e macrophages of the r e c i p i e n t mouse . A n t i b o d y - p r o d u c i n g h y b r i d c e l l s at a d e n s i t y of about 1 0 6 / n u in s e r u m f r e e RPMI 1640 medium were i n j e c t e d I.P. (1 ml/mouse) . S e v e n t o t e n d a y s a f t e r i n j e c t i o n wi th hybr idoma c e l l s , a s c i t e s f l u i d s were drawn from the abdominal c a v i t y . The drawn a s c i t e s f l u i d s were spun down a t 1500 rpm f o r 10 min t o p r e c i p i t a t e d e b r i s and r e d b l o o d c e l l s . The s u p e r n a t a n t s were p o o l e d t o g e t h e r and s t o r e d at - 2 0 ° C . The t i t e r of a n t i b o d i e s in the a s c i t e s f l u i d p r e p a r a t i o n was d e t e r m i n e d by t h e i n d i r e c t immunofluor e s c e n t a s s a y . P u r i f i c a t i o n of M o n o c l o n a l A n t i b o d i e s i n A s c i t e s F l u i d M o n o c l o n a l s p e r m a n t i b o d i e s were p u r i f i e d f rom a s c i t e s f l u i d a c c o r d i n g t o t h e s t a n d a r d p r o c e d u r e s [Chow et a l , 1985]. To 5 ml a s c i t e s f l u i d , 1.5 g of ammonium s u l f a t e was added t o b r i n g about 47% s a t u r a t i o n . A f t e r t h e p r e c i p i t a t e - 18 -was formed, i t was c e n t r i f u g e d at 27,OOOXg f o r 10 min at 4 ° C . The s u p e r n a t a n t was d i s c a r d e d , and the p e l l e t was r e s u s p e n d e d wi th d i s t i l l e d w a t e r t o t h e o r i g i n a l volume of a s c i t e s . The a b o v e d e s c r i b e d p r o c e d u r e was r e p e a t e d f o r the s e c o n d ammonium s u l f a t e f r a c t i o n a t i o n . A d i e t h y l a m i n o e t h y l (DEAE) S e p h a r o s e column was r e g e n e r a t e d wi th 50 ml of 0.5 M p o t a s s i u m p h o s p h a t e b u f f e r c o n t a i n i n g 6 M u r e a at pH 7.1. Then the b u f f e r was s w i t c h e d t o 10 mM p o t a s s i u m p h o s p h a t e at pH 7.1, and a g a i n t h e column was r e g e n e r a t e d wi th 100 ml of 10 mM b u f f e r at pH 7.1. A f t e r t h e s e c o n d ammonium s u l f a t e f r a c t i o n a t i o n , t h e p e l l e t was r e s u s p e n d e d in 5 ml d i s t i l l e d w a t e r , l o a d e d on the DEAE column and e l u t e d w i th 10 mM b u f f e r . F r a c t i o n s of 3 ml were c o l l e c t e d . O p t i c a l d e n s i t y (O.D.) was m e a s u r e d a t 280 nm, and t h e peak f r a c t i o n s were l o c a t e d and p o o l e d . They were a s s a y e d by the i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y u s i n g m e t h a n o l - f i x e d sperm t o c o n f i r m the e x i s t e n c e of sperm r e a c t i v e monoc lona l a n t i b o d y . By u s i n g SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s f o r m o l e c u l a r a n a l y s i s , t h e p u r i t y of the p u r i f i e d a n t i b o d i e s (in terms of IgG) r a n g e d f rom 70-90%. I n d i r e c t Immunof luorescent A s s a y and I n d i r e c t  Immunof luorescent I n h i b i t i o n A s s a y The method of i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y was d e s c r i b e d p r e v i o u s l y [Lee et a l , 1984a] . In t h i s a s s a y , the - 19 -s p e r m monoc lona l a n t i b o d i e s t o be t e s t e d s e r v e d as the f i r s t a n t i b o d y , and F I T C - l a b e l e d goat a n t i - m o u s e IgG+M+A as t h e s e c o n d a n t i b o d y . The p r e s e n c e of s p e c i f i c sperm ant igen( s ) in t e s t i s homogenate or in the p u r i f i e d p r e p a r a t i o n was d e t e c t e d by i t s a b i l i t y t o i n h i b i t immunofluor e s c e n t s t a i n i n g of sperm by a ^ g i v e n monoc lona l a n t i b o d y . To s t a n d a r d i z e the I n h i b i t i o n a s s a y p r o c e d u r e , t h e a n t i b o d y was d i l u t e d t o i t s end po in t of p o s i t i v e s t a i n i n g b e f o r e an i n h i b i t i o n a s s a y was p e r f o r m e d . To p e r f o r m t h e i n h i b i t i o n a s s a y , 50 u l of a n t i g e n s o l u t i o n was i n c u b a t e d wi th 50 u l of monoc lona l a n t i b o d y at i t s end po in t d i l u t i o n o v e r n i g h t at 4 ° C . The s o l u t i o n was t h e n i n c u b a t e d wi th m e t h a n o l - f i x e d mouse sperm on s l i d e s f o r one h o u r at room t e m p e r a t u r e . F o l l o w i n g t h r e e washes wi th P B S - B S A , F I T C - l a b e l e d goat a n t i - m o u s e IgG+M+A of known d i l u t i o n was added as t h e s e c o n d a n t i b o d y f o r an a d d i t i o n a l 40 minutes of i n c u b a t i o n a t room t e m p e r a t u r e . A f t e r t h r e e washes wi th P B S - B S A t o remove n o n - s p e c i f i c s t a i n i n g , the a b s e n c e of i n d i r e c t i m m u n o f l u o r e s c e n t s t a i n i n g of s p e r m a t o z o a would i n d i c a t e the p r e s e n c e of s p e r m ant igen( s ) i n a p a r t i c u l a r p r e p a r a t i o n . T e s t i s homogenate s e r v e d as the p o s i t i v e c o n t r o l , and l i v e r homogenate and a n t i g e n p r e p a r a t i o n wi th i r r e l e v a n t monoc lona l a n t i b o d y s e r v e d as the n e g a t i v e c o n t r o l s . E n z y m e - L i n k e d Immunosorbent A s s a y (ELISA) - 20 -ELISA was performed according to the procedures described previously [Voller et al, 1979; Lee et al, 1984c]. To examine the specificity of affinity-purified sperm antigens, the purified antigens were coated on microtiter plates for ELISA. A binding assay was performed by first incubating antibodies of various dilutions followed by washing and then the addition of goat anti-mouse Ig labeled with horse radish peroxidase. The enzymatic reaction was initiated by adding the substrates (pH 5.0 citrate buffer containing 0.2% OPD and 0.02% H 20 2) to determine the specificity of purified antigens and their cross-reactivity with different mnonoclonal sperm antibodies. Immuno-Afflnity Chromatography The sperm antigen reactive to MS 207 was purified by immuno-affinity chromatography using the monoclonal antibody as the affinity ligand. The purified antibody was coupled to Sepharose 4B with cyanogen bromide activation [Axen et al, 1967]. Generally 10 mg of the purified monoclonal antibody was coupled to 10 ml of Sepharose 4B. Following coupling, the immunoaffinity gel was washed three times with PBS and then incubated with 1 M Tris-HCL, pH 8.0 for one hour at room temperature to block the remaining sites for protein binding. Mouse testes were used as starting material to purify s p e r m a n t i g e n r e a c t i v e wi th MS 207. Mouse t e s t e s (3 g) were f i r s t homogenized in 10 ml of 50 mM T r i s - H C L , pH 8.0 c o n t a i n i n g 1 mM p h e n y l n e t h y l s u l f o n y l f l u o r i d e (PMSF) f o r one h o u r t o e x t r a c t s o l u b l e sperm a n t i g e n s . F o l l o w i n g c e n t r i f u g a t i o n a t 27,000Xg f o r 30 min, the s u p e r n a t a n t was d i l u t e d t o 1 O.D./ml wi th 50 mM t r i s - H C l (about 100 ml in volume). Th i s s o l u b l e f r a c t i o n of the mouse t e s t i s was t h e n l o a d e d on the MS 207 immunoaf f in i ty column (10 ml). The l o a d i n g was c a r r i e d out by c i r c u l a t i n g the homogenate t h r o u g h the column s l o w l y (flow r a t e = 0.5 ml/min) f o r 3 hr at room t e m p e r a t u r e . A f t e r l o a d i n g , t h e a f f i n i t y column was washed wi th 50 mM T r i s - H C l , pH 8.5 c o n t a i n i n g 0.5 M NaCl u n t i l t h e a b s o r b a n c e of O.D.280 d r o p p e d t o 0.002 or l e s s . The e l u t i o n was made wi th 50 mM d i e t h y l a m l n e b u f f e r at pH 10.0 t o f u r t h e r remove n o n s p e c i f i c m a t e r i a l s f r o m t h e column. Then the pH was s w i t c h e d t o 11.5. F r a c t i o n s c o n t a i n i n g OD.280 were c o l l e c t e d and n e u t r a l i z e d immediate ly wi th 0.5 M p h o s p h a t e b u f f e r (pH 7.3). To a c h i e v e h igher p u r i t y , t h e a n t i g e n - c o n t a i n i n g e l u e n t was d l a l y z e d a g a i n s t PBS o v e r night wi th 3 changes and t h e n l o a d e d o n t o the same a f f i n i t y column a g a i n . F o l l o w i n g t h e same p r o c e d u r e s of l o a d i n g and washing, the a n t i g e n was f i n a l l y o b t a i n e d in the e l u e n t of pH 11.5, which was immediate ly n e u t r a l i z e d with 0.5 M p h o s p h a t e b u f f e r , pH 7.3. - 22 -B e c a u s e the l e a k a g e of i m m u n o - a f f i n i t y l i g a n d , a n t i - s p e r m a n t i b o d y MS 207, was c o n s t a n t l y found in t h e e l u e n t of pH 11.5, an a f f i n i t y c h r o m a t o g r a p h y wi th r a b b i t a n t i - F c fragment of mouse IgG as a l i g a n d was c a r r i e d out t o remove the contaminant IgG from the e l u e n t . The o b t a i n e d mouse sperm a n t i g e n was named as M(ouse)S(perm)A(ntigen) 207. The i n d i r e c t i m m u n o f l u o r e s c e n t i n h i b i t i o n a s s a y was u s e d t o f o l l o w the a n t i g e n i c a c t i v i t y of the t a r g e t a n t i g e n . The f o l d of p u r i f i c a t i o n was e x p r e s s e d by " s p e c i f i c a c t i v i t y " , which was d e f i n e d as t h e i n v e r s e of p r o t e i n a b s o r b a n c e at 280 nm t h a t i s r e q u i r e d t o i n h i b i t MS 207 a t i t s end po in t d i l u t i o n i n i n d i r e c t t h e i m m u n o f l u o r e s c e n t a s s a y . The y i e l d of p u r i f i c a t i o n was e x p r e s s e d by " t o t a l a c t i v i t y " , i .e . , one a c t i v i t y e q u a l s t h e minimum O.D. e x h i b i t i n g i n h i b i t i o n on the b ind ing of MS 207 t o mouse sperm in t h e i m m u n o f l u o r e s c e n t a s s a y . Sodium D o d e c y l S u l f a t e - P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s F o l l o w i n g e a c h a n t i g e n p u r i f i c a t i o n , the a n t i g e n i c s p e c i f i c i t y of the a f f i n i t y - p u r i f i e d sperm a n t i g e n s was a n a l y z e d by i n d i r e c t immunof luorescent i n h i b i t i o n a s s a y d e s c r i b e d as a b o v e . The p u r i t y and t h e m o l e c u l a r weights of the p u r i f i e d a n t i g e n s were d e t e r m i n e d by sodium d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( S D S - P A G E , 6% a c r y l a m i d e gel) [Laemmli, 1970]. B r i e f l y , two c l e a n g l a s s - 23 -p l a t e s and two s p a c e r s were a s s e m b l e d and he ld by c l i p s . 6% g e l m i x t u r e was p r e p a r e d and d e g a s s e d . TEMED (25 u l i n 30 ml gel ) and p o t a s l u m p e r s u l f a t e (0.5 ml of 2% s t o c k in 30 ml gel ) were added . The mix ture was p i p e t t e d i n t o the chamber. The a c r y l a m i d e s o l u t i o n was t h e n o v e r l a y e r e d c a r e f u l l y wi th w a t e r t o e l i m i n a t e oxygen and t o g e n e r a t e a f l a t t o p t o t h e g e l . The s t a c k i n g g e l was t h e n p r e p a r e d and p o u r e d i n t o the gap remain ing i n the chamber . B e f o r e e l e c t r o p h o r e s i s , t h e samples were mixed wi th sample b u f f e r (10% SDS, 20% s u c r o s e and 10% a l p h a - t h i o g y c e r o l ) . The a p p a r a t u s was f i l l e d wi th r e s e r v o i r b u f f e r and the samples were i n j e c t e d i n t o the sample w e l l s . V o l t a g e s of 150 V and 250 V were a p p l i e d when the bromopheno l b l u e dye f r o n t was in the s t a c k i n g g e l and t h e 6% a c r y l a m i d e r u n n i n g g e l r e p e c t i v e l y [Smith, 1984]. A p p r o p r i a t e m o l e c u l a r s t a n d a r d s were u s e d f o r ge l e l e c t r o p h o r e s i s , i n c l u d i n g t h y r o g l o b u l i n (M.W. 670,000), gamma g l o b u l i n (M.W. 158,000), ovalbumin (M.W. 44,000) and myoglobin (M.W. 17,000) e t c . The a c r y l a m i d e g e l was t h e n s t a i n e d by a s i l v e r s t a i n i n g method [ M e r r i l et a l , 1981]. P r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d by L o w r y method [1951]. C a r b o h y d r a t e c o n t e n t of the p u r i f i e d sperm a n t i g e n s was d e t e r m i n e d by t h e p h e n o l - s u l f u r i c a c i d method of Hodge and H o f r e i t e r [1962] wi th s u c r o s e as the s u g a r s t a n d a r d . High P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y (HPLC) The f o l l o w i n g equipment were u s e d f o r HPLC: pump:BioRad - 24 -model 1330; m o n i t o r : B ioRad u.v. moni tor model 1306; r e c o r d e r : F i s h e r R e c o r d a l l 5000; column: B i o R a d HPLC g e l f i l t r a t i o n column B i o - S i l T S K - 2 5 0 (300 X 7.5 mm). The f low r a t e was 1 ml/min. F i f t y mM sodium p h o s p h a t e b u f f e r , pH6.7 c o n t a i n i n g 0.3 M NaCI was u s e d as r u n n i n g b u f f e r . A sample of 200 u l , which had b e e n d i a l y z e d a g a i n s t the same b u f f e r , was i n j e c t e d [Mayes , 1984]. F r a c t i o n s of 0.5 ml were c o l l e c t e d and t e s t e d in i m m u n o f l u o r e s c e n t i n h i b i t i o n a s s a y t o d e t e r m i n e the a c t i v e f r a c t i o n s . The p r e p a r a t i o n s were f r e e z e - d r i e d and s t o r e d at - 2 0 ° C u n t i l u s e . A c t i v e Immunization w i th P u r i f i e d Sperm A n t i g e n s Two g r o u p s of f o u r a d u l t female mice of C D - I s t r a i n were immunized wi th M S A - 2 0 7 as a t e s t and a d j u v a n t as a c o n t r o l , r e s p e c t i v e l y . A t y p i c a l animal immunizat ion reg imen was p e r f o r m e d . B r i e f l y , 20 ug of p u r i f i e d a n t i g e n s in 100 u l PBS was e m u l s i f i e d wi th an e q u a l volume of comple te F r e u n d ' s a d j u v a n t and i n j e c t e d s u b c u t a n e o u s l y t o e a c h mouse f o r the p r i m a r y immunizat ion . Subsequent immunizat ion was p e r f o r m e d a t t w o - w e e k i n t e r v a l s a f t e r t h e i n i t i a l one, except wi th incomple te F r e u n d ' s a d j u v a n t . S e v e n days a f t e r e a c h immunizat ion , animals were b l e d and t h e a n t i s e r u m t i t e r s were d e t e r m i n e d by i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y u s i n g m e t h a n o l - f i x e d mouse or human s p e r m a t o z o a [Lee et a l , 1984c] . - 25 -To d e t e r m i n e the s p e c i f i c i t y of t h e a n t i g e n - a n t i b o d y r e a c t i o n s , E L I S A [ V o l l e r et a l , 1979; L e e et a l , 1984c] and t h e double immunodi f fus ion on g e l was p e r f o r m e d a c c o r d i n g t o t h e p r o c e d u r e of O u c h t e r l o n y [1958]. A n t i f e r t i l i t y S t u d i e s A l l the r a i s e d a n t i s e r a were h e a t - i n a c t i v a t e d at 5 6 ° C f o r 45 min p r i o r t o use f o r a n t i f e r t i l i t y s t u d i e s . The a n t i f e r t i l i t y e f f e c t s of r a i s e d mouse isoimmune s e r a were e v a l u a t e d by t h e mouse in v i t r o f e r t i l i z a t i o n experiment and by t h e human sperm p e n e t r a t i o n a s s a y wi th z o n a - f r e e h a m s t e r o v a . The mouse in v i t r o f e r t i l i z a t i o n exper iments were p e r f o r m e d a c c o r d i n g t o p r e v i o u s l y r e p o r t e d p r o c e d u r e s [Biggers et a l , 1971, L e e et a l , 1986b]. B r i e f l y , female C D - I mouse was i n j e c t e d I.P. with 10 I.U. p r e g n a n t m a r e s ' s e r u m g o n a d o t r o p i n (PMSG) on day 1 and 10 I.U. hCH on day 3 t o Induce s u p e r o v u l a t i o n . S i x t e e n h o u r s a f t e r hCG i n j e c t i o n , masses of ova were r e c o v e r e d f r o m t h e ampul la r e g i o n of the o v i d u c t and p l a c e d in BWW medium [Biggers et a l , 1971]. Mouse sperm were t a k e n f rom the c a u d a epid idymis of two a d u l t male C D - I mice. The sperm were i n c u b a t e d in BWW medium at about 3 7 ° C f o r 10 min t o c a p a c i t a t e . The c o u n t s were t h e n a d j u s t e d t o 1 0 6 / m l by a p p r o p i a t e d i l u t i o n s . F o r t y - 26 -u l of the sperm s u s p e n s i o n was s u b s e q u e s t l y added t o 140 u l of BWW medium i n a p e t r i d i s h c o n t a i n i n g about 3.5 ml of p a r a f f i n o i l . Twenty u l of a n t i s e r u m or c o n t r o l s e r u m t o be t e s t e d were t h e n added i n t o the d r o p under t h e p a r a f f i n o i l in the p e t r i d i s h . The a n t i s e r a and the sperm were i n c u b a t e d t o g e t h e r a t 3 7 ° C f o r 30 min. The ova were t h e n added i n t o the d r o p . T h e y were i n c u b a t e d at 3 7 ° C f o r 7 h r . A f t e r i n c u b a t i o n the ova were washed 3 t imes c o n t i n u o u s l y wi th PBS and t h e n f i xed wi th 2% g l u t a r a l d e h y d e in PBS at 4 ° C o v e r n i g h t . The f i x e d ova were washed t w i c e wi th s a l i n e and t h e n s t a i n e d wi th 0.25% lacmoid in 45% a c e t i c a c i d f o r 10 t o 30 min. They were t h e n d e c o l o r i z e d wi th 20% g l y c e r o l p l u s 20% a c e t i c a c i d t w i c e . The ova were t r a n f e r r e d onto s l i d e s and o b s e r v e d under a m i c r o s c o p e . The c r i t e r i a f o r f e r t i l i z e d o o c y t e s were i d e n f i c a t i o n of : 1) b o t h male and female p r o n u c l e i , 2) b o t h f i r s t and s e c o n d p o l a r b o d i e s , and 3) the t a i l or whole segments of a sperm in the ovum. The sperm p e n e t r a t i o n a s s a y u s i n g z o n a - f r e e h a m s t e r ova and human s p e r m a t o z o a was p e r f o r m e d a c c o r d i n g t o the p r o c e d u r e of Yanagimachl et a l [1976]. B r i e f l y , semen f r o m males of p r o v e n f e r t i l i t y was g e n t l y added at the b o t t o m of a t u b e c o n t a i n i n g 5 ml BWW medium. The t u b e was t h e n i n c u b a t e d at 3 7 ° C t o a l l o w mot i l e sperm t o swim up t o the medium p h a s e . The s p e r m s u s p e n s i o n i n BWW medium was i n c u b a t e d a t 37 °C o v e r n i g h t and c o u n t s a d j u s t e d t o 10 . 6 /ml . Adul t female h a m s t e r s of L V G s t r a i n were i n j e c t e d wi th 40 I.U of PMSG on day 1 and 40 I.U. of hCG on day 3. S i x t e e n h o u r s a f t e r hCG i n j e c t i o n , the ova were r e c o v e r e d f rom the o v i d u c t s and i n c u b a t e d wi th h y a l u r o n i d a s e (0.1% in BWW medium) at room t e m p e r a t u r e t o remove cumulus c e l l s . A f t e r washing wi th BWW, t h e ova were i n c u b a t e d w i th 0.1% t r y p s i n t o remove zona p e l l u c e d a e . F o r t y u l of human s p e r m s u s p e n s i o n was added in a p e t r i d i s h c o n t a i n i n g a d r o p of 140 u l of BWW, and 20 u l of i n a c t i v a t e d a n t i s e r a or c o n t r o l s e r a t h a t were t o be t e s t e d i n p a r a f f i n o i l . They were i n c u b a t e d at 3 7 ° C f o r 30 min. The z o n a - f r e e h a m s t e r ova were t h e n added i n t o the d r o p and i n c u b a t e d a t 37 "C f o r 3 h r . A f t e r i n c u b a t i o n the o v a were washed 3 t imes wi th PBS and t r a n s f e r r e d onto a s l i d e and o b s e r v e d under p h a s e - c o n t r a s t m i c r o s c o p e . The same c r i t e r i a f o r f e r t i l i z e d ova as in the mouse in v i t r o f e r t i l i z a t i o n exper iment were a p p l i e d . A d e q u a t e c o n t r o l s were i n c l u d e d i n e a c h experiment s u c h as normal mouse s e r a , N S - 1 a s c i t e s f l u i d , a n t i s e r a a g a i n s t u n r e l a t e d p r o t e i n s and monoc lona l a n t i b o d i e s a g a i n s t sperm or u n r e l a t e d p r o t e i n s . S t a t i s t i c a l A n a l y s i s C h i - s q u a r e method was u s e d t o a n a l y z e f o r t h e r • s i g n i f i c a n c e of d i f f e r e n c e s . - 28 -R E S U L T S A n t i g e n P u r i f i c a t i o n A f t e r c y a n o g e n bromide a c t i v a t i o n , the p u r i f i e d monoc lona l a n t i b o d y , MS 207 was immobil ized on S e p h a r o s e 4B, as judged f r o m t h e s i g n i f i c a n t d e c r e a s e in O.D.280 r e a d i n g i n the s o l u t i o n r e c o v e r e d f rom the c o u p l i n g as compared wi th the o r i g i n a l . G e n e r a l l y a b o u t 80 - 95% of the o r i g i n a l p r o t e i n was c o v a l e n t l y bound t o the g e l . The i n h i b i t o r y a c t i v i t y of t h e sperm a n t i g e n was only found in the s o l u b l e f r a c t i o n of t h e mouse t e s t i s homogenate . As shown by t h e i n d i r e c t i m m u n o f l u o r e s c e n t i n h i b i t i o n a s s a y , a f t e r e x t e n s i v e washes of the p e l l e t of the t e s t i s homogenate w i th 50 mM t r i s - H C l , t h e d e t e r g e n t - s o l u b l i z e d f r a c t i o n d id not exhib i t any I n h i b i t o r y a c t i v i t y . F r o m the s o l u b l e f r a c t i o n of t h e t e s t i s homogenate , t h e mouse s p e r m a n t i g e n r e a c t i v e wi th MS 207 was s u b s t a n t i a l l y p u r i f i e d by i m m u n o - a f f i n i t y c h r o m a t o g r a p h y . In o r d e r t o a c h i e v e h i g h e r p u r i t y , a s e c o n d p u r i f i c a t i o n was c a r r i e d out by t h e same column. One of t h e t y p i c a l e l u t i o n p r o f i l e s i s p r e s e n t e d in F i g 1. - 29 -The i n h i b i t o r y a c t i v i t y in e a c h s t e p s of the p u r i f i c a t i o n was f o l l o w e d by i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y , so t h a t the t o t a l and s p e c i f i c a c t i v i t y c o u l d be c a l c u l a t e d (Fig 2, Tab I). A f t e r two a f f i n i t y c h r o m a t o g r a p h i c p u r i f i c a t i o n , the s p e c i f i c a c t i v i t y i n c r e a c e d 2,700 f o l d s i n t h e f i n a l p r e p a r a t i o n as compared wi th the c r u d e homogenate . Howeveer , a f t e r the p u r i f i c a t i o n , the t o t a l a c t i v i t y r e c o v e r e d was only a b o u t 20% (Fig 2). F o l l o w i n g the c o a t i n g of each of the p u r i f i e d a n t i g e n s on micro t i t e r p l a t e s , E L I S A was a l s o p e r f o r m e d t o d e t e c t t h e b ind ing of MS 207 t o MSA 207. S imi lar r e s u l t s were o b t a i n e d as shown in i m m u n o f l u o r e s c e n t a s s a y s ; the c o a t e d M S A - 2 0 7 only r e a c t e d wi th MS 207, but not wi th o t h e r u n r e l a t e d monoc lona l a n t i - s p e r m a n t i b o d i e s (F ig 3). The p u r i f i e d a n t i g e n s were t h e n a n a l y z e d u s i n g SDS a c r y l a m i d e g e l e l e c t r o p h o r e s i s (6% a c r y l a m i d e gel ) . The a n t i g e n s r e a c t i v e wi th MS 207 were d e t e r m i n e d t o have m o l e c u l a r weights of 600 - 700 KDa in t h e p r e s e n c e of a r e d u c i n g agent (F ig 4). The sample c o n t a i n i n g the p u r i f i e d M S A - 2 0 7 was f u r t h e r a n a l y z e d by H P L C - g e l f i l t r a t i o n . The p r o f i l e of O.D.280 Is p r e s e n t e d in F i g 5. The major peak was found at t h e f r a c t i o n of m o l e c u l a r weight of a b o u t 600 KDa . Thi s peak was found t o e l i c i t the a n t i g e n i c a c t i v i t y t o i n h i b i t the b ind ing of MS 207 t o the mouse sperm as o b s e r v e d - 30 -in i m m u n o f l u o r e s c e n t i n h i b i t i o n a s s a y s . The p u r i f i e d sperm a n t i g e n was shown t o be a g l y c o p r o t e i n with the c a r b o h y d r a t e c o n t e n t of 20%. A c t i v e Immunization wi th P u r i f i e d A n t i g e n s Female mice were immunized wi th M S A - 2 0 7 t o t e s t the immunogenic i ty and a n t i f e r t i l i t y e f f e c t of a c t i v e immunizat ion . A f t e r s u c c e s s i v e immunizat ion, t h e animals were b l e d and a n t i b o d y t i t e r s were d e t e r m i n e d by t h e i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y u s i n g methano l f i xed human or mouse s p e r m (F ig 6). F i g u r e 7 shows t h a t t h e a n t i b o d y t i t e r s a p p e a r e d t o i n c r e a s e with i n c r e a s i n g number of immunizat ion . A f t e r t h e f o u t h immunizat ion the a n t i b o d y t i t e r s from t h e mice immunized wi th M S A - 2 0 7 were found t o be 1:800. The r a i s e d mouse s e r u m were shown t o c r o s s - r e a c t w i th s p e r m from mouse and human. I n t e r e s t i n g l y , whereas MS 207 is a mouse sperm s p e c i f i c monoc lona l a n t i b o d y , a n t i s e r a a g a i n s t M S A - 2 0 7 showed c r o s s r e a c t i v i t y wi th human s p e r m (F ig 6). The a n t i s e r a bound t o the s i m i l a r r e g i o n as t h e i r c o r r e s p o n d i n g monoc lona l a n t i b o d i e s . None of the a n t i s e r a was found t o r e a c t wi th o t h e r s o m a t i c o r g a n s , s u c h as b r a i n , s p l e e n , l i v e r , k idney or h e a r t as judged by i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y s and t h e - 31 -O u c h t e r l o n y double immunodi f fus ion t e c h n i q u e . In V i t r o F e r t i l i z a t i o n Exper iment and Sperm P e n e t r a t i o n A s s a y The a n t i f e r t i l i t y e f f e c t s of the r a i s e d isoimmune s e r a were e v a l u a t e d by t h e i n v i t r o f e r t i l i z a t i o n experiment (mouse ovum/mouse sperm) and the sperm p e n e t r a t i o n a s s a y ( z o n a - f r e e h a m s t e r ovum/human sperm). The r e s u l t s of t h e s e a n a l y s e s a r e p r e s e n t e d in T a b l e II and III. In p r e s e n c e of a n t i - M S A - 2 0 7 , t h e f e r t i l i z a t i o n r a t e of mouse o v a was as low as 8.3%, while a 67.7% f e r t i l i z a t i o n r a t e was o b s e r v e d in t h e c o n t r o l s e r u m . The monoc lona l a n t i - s p e r m a n t i b o d y , MS 207, showed s i m i l a r a n t i - f e r t i l i z a t i o n e f f e c t (Table II). The a n t i s e r a a l s o showed v e r y s t r o n g i n h i b i t i o n on human sperm p e n e t r a t i o n of z o n a - f r e e h a m s t e r ova (4.1%) as compared t o t h e c o n t r o l s e r u m (62.2%, T a b l e III). - 32 -DISCUSSION The r e s e a r c h and deve lopment of c o n t r a c e p t i v e v a c c i n e s i s a l o n g t e r m m i s s i o n of the A n d r o l o g y L a b o r a t o r y at UBC. Much p r e l i m i n a r y work has b e e n done by o t h e r w o r k e r s i n t h i s l a b o r a t o r y s i n c e 1982. It was o b s e r v e d t h a t MS 207, a mouse sperm s p e c i f i c monoc lona l a n t i b o d y , b inds t o t h e a n t e r i o r a c r o s o m e of mouse sperm and a f f e c t s s i g n i f i c a n t l y the f e r t i l i z a t i o n of mouse ova i n v i t r o and in v i v o [Lee et a l , 1986]. The mechanism by which MS 207 i n t e r f e r e s wi th f e r t i l i z a t i o n might induce t h e a c r o s o m a l r e a c t i o n s , which a r e e s s e n t i a l f o r s p e r m p e n e t r a t i o n t h r o u g h the zona p e l l u c l d a [Lee et a l , 1987]. In t h i s t h e s i s work, MS 207 was c h o s e n t o p u r i f y mouse s p e r m a n t i g e n b e c a u s e i t e x h i b i t s a s t r o n g a n t i f e r t i l i t y e f f e c t b o t h in v i t r o and i n v i v o [Lee , et a l , 1987]. It was s p e c u l a t e d t h a t the c o r r e s p o n d i n g a n t i g e n t o MS 207 might be a i m p o r t a n t f a c t o r in f e r t i l i z a t i o n p r o c e s s . P u r i f i c a t i o n of Sperm A n t i g e n s U s i n g monoc lona l sperm a n t i b o d i e s t o p u r i f y sperm a n t i g e n s is the b e s t a p p r o a c h t o c a r r y out s t u d i e s in immunolog ica l f e r t i l i t y c o n t r o l . F i r s t of a l l , by c h o o s i n g s p e r m - s p e c i f i c monoc lona l a n t i b o d i e s , the c o r r e s p o n d i n g - 3 3 -a n t i g e n s would not l i k e l y induce immune r e s p o n s e s t h a t c r o s s r e a c t w i th a n t i g e n s p r e s e n t in o t h e r t i s s u e s . T h i s i s t h e unique a d v a n t a g e of the s i n g l e - d e t e r m i n a n t - s p e c i f i c i t y of monoc lona l a n t i b o d i e s . S e c o n d l y , i t has b e e n r e p o r t e d t h a t the p r e s e n c e of numerous a n t i g e n s in t h e whole e x t r a c t may modula te t h e immunogenic i ty of some o t h e r a n t i g e n i c p r o t e i n s r e l e v a n t t o f e r t i l i t y . F o r example, immunizat ion of mice wi th i s o l o g o u s i n t a c t sperm l e a d s t o the f o r m a t i o n of a n t i s p e r m a n t i b o d i e s r e a c t i n g a g a i n s t v a r i o u s a n t i g e n s but not a g a i n s t weak immunogens s u c h as L D H - C 4 [Tung et a l , 1979]. Immunization wi th h e t e r o l o g o u s or i s o l o g o u s i n t a c t zona p e l u c i d a r e s u l t s i n t h e f o r m a t i o n of a n t i z o n a a n t i b o d i e s which do not r e a c t wi th sperm r e c e p t o r on t h e zona p e l u c i d a [Ahuja and T z a r t o s , 1981] . T h e r e f o r e immunizat ion wi th p u r i f i e d s i n g l e a n t i g e n might r e d u c e f e r t i l i t y more d r a m a t i c a l l y t h a n t h a t by immunizat ion wi th mix ture of a n t i g e n s . Numerous methods a r e a v a i l a b l e f o r p r o t e i n p u r i f i c a t i o n . They i n c l u d e ion exchange c h r o m a t o g r a p h y , g e l f i l t r a t i o n , e l e c t r o p h o r e s i s , e t c . T h e s e methods a r e b a s e d on the d i f f e r e n c e s in the p h y s i c a l and chemica l p r o p e r t i e s of p r o t e i n m i x t u r e s . By t h e s e c o n v e n t i o n a l methods , the s p e c i f i c i t y i s low and m u l t i p l e s t e p s a r e r e q u i r e d t o a c h i e v e t h e d e s i r e d p u r i t y . Compared wi th t h e a b o v e t i m e - c o n s u m i n g methods , a f f i n i t y c h r o m a t o g r a p h y is t h e most e f f e c t i v e method f o r p u r i f y i n g p r o t e i n s . T h i s method i s b a s e d on the unique binding specificity of biological macromolecules. For example, the antigen-antibody binding in immune reactions is highly specific. Therefore monoclonal antibodies provide a good ligand to purify membrane proteins [Azzi et al, 1981]. By means of immunoaffinity chromatography with a monoclonal antibody as the ligand, the sperm antigen reactive with MS 207 was purified from mouse testis. It was found that the antigenic activity only existed in the soluble fraction of the the mouse testis homogenate. A detergent, deoxycholate, could not extract anything active after the membrane fraction (pellet) of the homogenate was extensively washed by a buffer not containing detergent. This finding suggests that the sperm antigen reactive with MS 207 (MSA-207) might not be a membrane bound protein, because most membrane bound proteins are insoluble without detergent [Lehninger,1982]. However, MSA-207 is exposed to the outside of sperm membrane, and cannot be washed away as observed in immunofluorescent assay. This previous datum seems hard to explain; but similar findings were also observed by other authors. For example, LDH-C4 (LDH-X) is a soluble protein with an enzymatic activity. However, LDH-C4 can be detected on the pserm tail by indirect immunofluorescent assay even after the sperm is extensively washed by PBS [Goldburg, 1979]. This property of these proteins remains to be investigated. However, this observation does not affect the major objectives of this study. - 35 -By u s i n g d e s i g n e d i n d i r e c t i m m u n o f l u o r e s c e n t i n h i b i t i o n a s s a y and E L I S A p r o c e d u r e s , t h e b ind ing a c t i v i t y of the p u r i f i e d sperm a n t i g e n s t o the c o r r e s p o n d i n g monoc lona l a n t i b o d i e s c o u l d be f o l l o w e d . A f t e r a f f i n i t y c h r o m a t o g r a p h i c p u r i f i c a t i o n , the e l u t e d sperm a n t i g e n s r e t a i n e d t h e i r a n t i b o d y b ind ing a c t i v i t y a t n e u t r a l pH. O b v i o u s e l y , the only way t o t r a c e the a c t i v i t y of t h i s a n t i g e n is t o f o l l o w i t s a n t i g e n i c a c t i v i t y . The s p e c i f i c a c t i v i t y ( fo lds of the p u r i f i c a t i o n ) and the t o t a l a c t i v i t y ( y i e l d of the p u r i f i c a t i o n ) a r e p r e s e n t e d in Tab I. It i s shown t h a t more t h a n 4 0 0 - f o l d p u r i f i c a t i o n was a c h i e v e d a f t e r t h e f i r s t a f f i n i t y c h r o m a t o g r a p h i c p u r i f i c a t i o n . The n e c e s s i t y of t h e s e c o n d column p u r i f i c a t i o n is shown by the r e s u l t t h a t the p u r i f y i n c r e a s e d about 6 t imes w i thout l o s s o f the t o t a l a c t i v i t y . The y i e l d of the t o t a l a c t i v i t y a f t e r two p u r i f i c a t i o n s t e p i s r a t h e r low (about 20%). However , about h a l f of the t o t a l a c t i v i t y was l o s t in the p e l l e t of the t e s t i s homogenate (Tab I), a l t h o u g h i t was e x e n s i v e l y washed. The o t h e r major l o s s of a c t i v i t y i s f rom the f i r s t a f f i n i t y chromatography, which i s a p p a r e n t l y due t o t h e c a p a c i t y of t h e a f f i n i t y column (Tab I). The H P L C - g e l f i l t r a t i o n was c a r r i e d out t o a n a l y z e the m o l e c u l a r weight of the i m m u n o r e a c t i v e m o l e c u l e . As shown in F i g 5, t h e major peak (peak A) a p p e a r e d b e t w e e n 6 and 7.5 min, which i s the p o s i t i o n of m o l e c u l a r weight of 600 t o 700 - 36 -KDa. Only the samples c o l l e c t e d f rom t h i s peak e x h i b i t e d i m m u n o r e a c t i v i t y , c o n f i r m i n g t h e m o l e c u l a r weight of t h e t a r g e t m o l e c u l e . The peak B is a b u f f e r a r t i f a c t . The S D S - P A G E showed t h e s i m i l a r r e s u l t (F ig 4). Such a h i g h - m o l e c u l a r weight a n t i g e n s h o u l d be of m u l t i p l e - e p i t o p e . F u r t h e r s t u d i e s s h o u l d be d i r e c t e d t o the p o s s i b l e enzymat i c a c t i v i t y on the a n t i g e n molecu le and a n a l y s i s of the ep i t opes . A c t i v e Immunization of P u r i f i e d Sperm A n t i g e n s In Mouse F o r the p u r p o s e of i m m u n o c o n t r a c e p t i o n , p a s s i v e immunizat ion would not be p r a c t i c a l . The b i o l o g i c a l h a l f l i f e of IgG is a b o u t 14 d a y s . F r e q u e n t i n j e c t i o n s may be u n d e s i r a b l e in fami ly p l a n n i n g . A c t i v e immunizat ion wi th a n t i g e n s would e l i c i t a s t r o n g e r and l o n g e r e f f e c t t o body's immune s y s t e m [Alexander and A n d e r s o n , 1983]. Th i s s t r a t e g y is now c o n s i d e r e d as t h e only p o s s i b l e means in s p e r m - b a s e d i m m u n o c o n t r a c e p t i o n . F r o m t h e exper iments c o n d u c t e d p r e v i o u s l y by L e e and h i s c o - w o r k e r s , i t was shown t h a t p a s s i v e immunizat ion ln female mice wi th h igh d o s e of MS 207 (40 mg/kg body weight) e x h i b i t e d a s t r o n g i n h i b i t o r y e f f e c t on in v i t r o f e r t i l i z a t i o n of mouse o v a , and a p a r t i a l i n h i b i t o r y e f f e c t on in v i v o f e r t i l i z a t i o n r a t e s and s u b s e q u e n t embryo deve lopment in v i t r o [Lee et a l , 1987] . - 37 -When t h e a f f i n i t y - p u r i f i e d sperm antigen's were u s e d t o isoimmunize female mice, the a n t i s e r a were shown t o have comparab le a n t i b o d y t i t e r s and r e a c t e x c l u s i v e l y wi th t h e a c r o s o m e of s p e r m a t o z o a f rom human and mouse. It has been p r e v i o u s l y o b s e r v e d t h a t sperm monoc lona l a n t i b o d y , MS 207 i s mouse s p e c i f i c , w i thout c r o s s - r e a c t i v i t y wi th human s p e r m [Lee et a l , 1986]. However , u s i n g MS 207 as an a f f i n i t y l i g a n d , the p u r i f i e d a n t i g e n c o u l d e l i c i t immune r e s p o n s e t h a t c r o s s - r e a c t wi th sperm f r o m o t h e r s p e c i e s . Th i s i n d i c a t e s t h a t the isoimmune r e s p o n s e was a l s o induced t o c r o s s - r e a c t i v e e p i t o p e s of the a f f i n i t y p u r i f i e d a n t i g e n s i n o t h e r s p e c i e s . T h i s o b s e r v a t i o n s u g g e s t s a new s t r a t e g y f o r d e v e l o p i n g c o n t r a c e p t i v e v a c c i n e s . It was p r e v i o u s l y thought t h a t only monoc lona l a n t i b o d i e s a g a i n s t human s p e r m c o u l d be u s e d as p r o b e s t o o b t a i n a f f i n i t y - p u r i f i e d sperm a n t i g e n s f o r the p u r p o s e of human c o n t r a c e p t i o n . Now i t has been shown t o be p o s s i b l e t h a t a monoc lona l a n t i - s p e r m a n t i b o d y wi th no c r o s s - r e a c t i v i t y t o human sperm c a n a l s o be u s e d t o p u r i f y a n t i g e n s f r o m o t h e r s p e c i e s . The common e p i t o p e s i n t h e a n t i g e n i c molecu le might e x i s t in human sperm, and induce immune r e s p o n s e s a g a i n s t human sperm. However , s u c h an immunizat ion would be c o m p l i c a t e d and the s i d e e f f e c t s of h e t e r o l o g o u s immunizat ion s h o u l d be c o n s i d e r e d [Jones , 1982]. A n t i f e r t i l i t y E f f e c t  Sperm A n t i g e n s of A c t i v e Immunization wi th P u r i f i e d - 38 -The mouse in v i t r o f e r t i l i z a t i o n experiment was employed t o e v a l u a t e t h e a n t i f e r t i l i t y e f f e c t s of the a n t i s e r a r a i s e d by M S A - 2 0 7 immunizat ion . It was c l e a r l y d e m o n s t r a t e d t h a t t h e f e r t i l i z i n g f u n c t i o n o f mouse sperm was s t r o n g l y b l o c k e d by the a n t i s e r a (Table II). S i m i l a r l y , the a n t i s e r a a l s o showed a s t r o n g i n h i b i t i o n of the f e r t i l i z i n g a b i l i t y of human sperm as o b s e r v e d in the sperm p e n e t r a t i o n a s s a y u s i n g z o n a - f r e e h a m s t e r o v a . It has b e e n p r e v i o u s l y o b s e r v e d t h a t IgG i n the s o m a t i c c i r c u l a t i o n c a n e n t e r the female r e p r o d u c t i v e t r a c t a lmost f r e e l y . The l e v e l of IgG in the f l u i d of t h e female r e p r o d u c t i v e t r a c t , i n c l u d i n g v a g i n a , u t e r u s and o v i d u c t , i s a lmost same as t h a t i n t h e b l o o d [Jones , 1982]. Thus i t c o u l d be e x p e c t e d t h a t a comparab le a n t i f e r t i l i t y e f f e c t can be d e m o n s t r a t e d i n mat ing t e s t s as in i n v i t r o f u n c t i o n a l t e s t s shown a b o v e . T h i s , as a n o t h e r p a r t of the l o n g - t e r m p r o j e c t , i s now b e i n g c o n d u c t e d in C h i n a . - 39 -CONCLUSIONS T h e f o l l o w i n g c o n c l u s i o n s c a n b e d r a w n f r o m t h i s t h e s i s : 1. W i t h i m m u n o a f f i n i t y c h r o m a t o g r a p h y , s p e r m a c r o s o m a l a n t i g e n s r e a c t i v e w i t h MS 2 0 7 w e r e s u c c e s s f u l l y p u r i f i e d f r o m m o u s e t e s t e s . T h e a n t i g e n c a n b e f o u n d o n l y f r o m s o l u b l e f r a c t i o n o f t h e t e s t i s h o m o g e n a t e . It i s a g l y c o p r o t e i n w i t h m o l e c u l a r w e i g h t o f a b o u t 6 0 0 t o 7 0 0 K D a . T h i s p r o t e i n m a i n t a i n s i t s i m m u n o r e a c t i v i t y a f t e r t h e a f f i n i t y c h r o m a t o g r a p h i c p u r i f i c a t i o n . 2. A c t i v e i m m u n i z a t i o n i n m i c e w i t h M S A - 2 0 7 c o u l d e l i c i t a s t r o n g immune r e s p o n s e t o m o u s e a n d h u m a n s p e r m . B y i n v i t r o f u n c t i o n a l t e s t s , m o u s e s p e r m f e r t i l i z i n g a n d h u m a n s p e r m p e n e t r a t i n g f u n c t i o n s a r e a l m o s t c o m p l e t e l y b l o c k e d b y a n t i s e r a r a i s e d b y i s o - i m m u n i z a t i o n w i t h M S A - 2 0 7 . - 4 0 -PART TWO. STUDIES OF ANTI-IDIOTYPIC ANTIBODIES AGAINST A MONOCLONAL SPERM ANTIBODY MATERIALS AND METHODS Chemica l s P r o t e i n A, L a c m o i d , c y s t e i n e , A l p h a - t h i o g l y c e r o l and m e r c u r i p a p a i n were p u r c h a s e d f rom Sigma Chemica l Company, S t . L o u i s , MO.. M i c r o t i t e r p l a t e s were f rom FLOW l a b o r a t o r i e s , M i s s i s s a u g a , O n t a r i o . A l l o t h e r chemica l s have been d e s c r i b e d i n P a r t One. Animals Randomly b r e d C D - I mice were f r o m C h a r l e s R i v e r , Canada Inc.. Female New Z e a l a n d White r a b b i t s were p u r c h a s e d the f r o m Animal C a r e Unit of UBC. M o n o c l o n a l A n t i b o d y a g a i n s t Mouse Sperm — MS 204 MS 204 i s s e c r e t e d by a hybr idoma c e l l l i n e g e n e r a t e d by L e e and h i s c o - w o r k e r s [1984,1987] . T h i s a n t i b o d y r e a c t s - 41 -with the a c r o s o m a l r e g i o n of mouse s p e r m s p e c i f i c a l l y , w i thout c r o s s - r e a c t i v i t y with e i t h e r o t h e r s o m a t i c t i s s u e s or sperm from o t h e r s p e c i e s [Lee , et a l , 1986]. It e x h i b i t s s t r o n g i n h i b i t i o n of mouse s p e r m f e r t i l i z i n g c a p a c i t y b o t h in v i t r o and in v i v o [Lee et a l , 1986]. However , i t seems t h a t MS 204 r e a c t s wi th a c o n f o r m a t i o n a l a n t i g e n , which is l o s t a f t e r d e t e r g e n t e x a c t i o n [unpubl i shed o b s e r v a t i o n ] . S ince i t i s r e l a t i v e l y d i f f i c u l t t o p u r i f y the MS 2 0 4 - r e a c t i v e s p e r m a n t i g e n s , we d e c i d e d t o g e n e r a t e a n t i - i d i o t y p i c a n t i b o d i e s t o MS 204, which may a c t as t h e " i n t e r n a l image" of t h e c o r r e s p o n d i n g sperm sperm a n t i g e n s . Immunization wi th MS 204 ln Rabbit A t y p i c a l immunizat ion reg imen was p e r f o r m e d [Hood et a l , 1984]. B r i e f l y , 50 ug of MS 204 in 0.5 ml PBS was e m u l s i f i e d wi th an e q u a l volume of comple te F r e u n d ' s a d j u v a n t and i n j e c t d s u b c u t a n e o u s l y in one r a b b i t f o r p r i m a r y immunizat ion . S u b s e q u e n t immunizat ions were p e r f o r m e d at t w o - w e e k i n t e r v a l s a f t e r the i n i t i a l one, except with imcomplete F r e u n d ' s a d j u v a n t . B l o o d was drawn t h r o u g h t h e e a r v e i n , and s e r u m was o b t a i n e d by c e n t i f u g a t i o n , and was s t o r e d at - 2 0 ° C u n t i l u s e . P u r i f i c a t i o n of A n t i - i d i o t y p i c A n t i b o d y a g a i n s t MS 204 MS 204, GO 15, an i r r e l e v a n t monoc lona l a n t i b o d y a g a i n s t - 42 -hCG, and p r o t e i n A were c o u p l e d onto S e p h a r o s e 4B a c c o r d i n g s t a n d a r d p r o c e d u r e d e s c r i b e d p r e v i o u s l y [Axen et a l , 1967]. G e n e r a l l y 10 img o f p r o t e i n were u s e d t o c o u p l e 10 ml of S e p h a r o s e 4B g e l . F o l l o w i n g c o u p l i n g , t h e immunoaff i n i t y g e l was washed t h r e e t imes wi th PBS and t h e n i n c u b a t e d wi th 1 M T r i s - H C l , pH 8.0, f o r one h o u r a t room t e m p e r a t u r e t o b l o c k t h e remain ing s i t e s f o r p r o t e i n b ind ing . Rabbi t a n t i s e r u m a g a i n s t MS 204 (10 ml) was f i r s t p a s s e d t h r o u g h GO 15 column (10 ml) t o remove a n t i - F c f r a c t i o n f r o m t h e s e r u m . The s e r u m was a l l o w e d t o c i r c u l a t e t h r o u g h t h e column at the f low r a t e of 20 ml /hr f o r 30 min. The s e r u m was t h e n c o l l e c t e d and l o a d e d on MS 204 a f f i n i t y column. The GO 15 column was e l u t e d wi th 50 mM g l y c i n e - H C 1 , pH 2.8, and peak f r a c t i o n s of O.O.280 r e a d i n g were c o l l e c t e d and t e s t e d by r a d i o b i n d i n g a s s a y t o d e m o n s t r a t e the e x i s t a n c e of r a b b i t a n t i - m o u s e IgG [Lee et a l , 1986]. The same p r o c e d u r e was p e r f o r m e d f o r MS 204 a f f i n i t y c h r o m a t o g r a p h y as f o r GO 15. F o l l o w i n g 30 min l o a d i n g and e x t e n s i v e washing wi th 50 mM t r i s - H C l , pH 8.0, c o n t a i n i n g 0.5 M N a C l , the column was e l u t e d wi th g l y c i n e - H C l , pH 2.8. Peak f r a c t i o n s of O.D.280 were c o l l e c t e d and p o o l e d and n e u t r a l i z e d wi th s o l i d sodium b i c a r b o n a t e . P r o t e i n A a f f i n i t y column was u s e d t o f u r t h e r p u r i f y r a b b i t IgG in the p o o l f o l l o w i n g the same p r o c e d u r e as d e s c r i b e d a b o v e . The p u r i f i e d r a b b i t a n t i - F a b fragment of MS 204 - 43 -( A n t l - i d i o t y p e t o MS 204, A id 204) was examined wi th i n d i r e c t i m m u n o f l u o r e s c e n t i n h i b i t i o n a s s a y [see P a r t One of the t h e s i s ] . A i d 204 s h o u l d be a b l e t o i n h i b i t the b ind ing of MS 204 t o mouse sperm. Immunization wi th A i d 204 in female mice The p u r i f i e d Aid 204 was c o n j u g a t e d wi th hemocyanin a c c o r d i n g t o t h e s t a n d a r d p r o c e d u r e [Chow et a l , 1985]. F o r p r i m a r y immunizat ion , 20 ug of A id 204 in 100 u l PBS (exc luding c o n j u g a t e d hemocyanin) was e m u l s i f i e d w i th e q u a l volumn of F r u e d ' s comple te a d j u v a n t and i n j e c t e d s u b c u t a n e o u s e l y i n t o e a c h of 3 female C D - I mice. A n o t h e r g r o u p of 3 mice were i n j e c t e d wi th a d j u v a n t only as c o n t r o l . Subsequent immunizat ions were p e r f o r m e d at 2-week i n t e r v a l s in Fruend ' s Incomplete a d j u v a n t . Animals were b l e d and the a n t i s e r a t i t e r s were d e t e r m i n e d by E L I S A wi th m i c r o t i t e r p l a t e s c o a t e d wi th Fab fragment of A id 204. E n z y m e - L i n k e d Immunosorbent A s s a y (ELISA) E L I S A was p e r f o r m e d a c c o r d i n g t o p r o c e d u r e s d e s c r i b e d in P a r t One. F a b fragment of A i d 204 was c o a t e d on t h e m i c r o t i t e r p l a t e s . The Fab of A id 204 was o b t a i n e d by p a p a i n d i g e s t i o n [ G a r v e y et a l , 1977] . B r i e f l y , m e r c u r i p a p a i n was - 44 -added a t 1% (w/w) of r a b b i t immunoglobulin i n b u f f e r s o l u t i o n (0.1 M p h o s p h a t e b u f f e r , pH7.0; lOraM c y s t e i n e , 2mM EDTA). T h e y were t h e n i n c u b a t e d at 3 7 ° C f o r 7 h o u r s . The d i g e s t e d sample was d i a l y s e d f i r s t a g a i n s t d i s t i l l e d w a t e r f o r 48 h o u r s t o remove c y s t e i n e , t h e n d i l u t e d t o 3 ug/ml i n 50 mM T r i s - H C l , pH 7.5 f o r c o a t i n g . Double Immunodif fus ion, I n d i r e c t Immunof luorescent A s s a y and In V i t r o F e r t i l i z a t i o n T e s t Us ing Mouse Sperms and Ova were a l l p e r f o r m e d a c c o r d i n g t o t h e p r o c e d u r e s d e s c r i b e d in P a r t One. C h i - s q u a r e method was u s e d t o a n a l y z e the s i g n i f i c a n c e of d i f f e r e n c e s in t h e i n v i t r o f e r t i l i z a t i o n r a t e s . - 45 -R E S U L T S G e n e r a t i o n of Rabbi t A n t i s e r u m aga ln t MS 204 A f t e r s u c c e s s i v e immunizat ions , the a n t i s e r u m a g a i n s t monoc lona l sperm a n t i b o d y MS 204 was r a i s e d in one female r a b b i t as judged by double immunodi f fus ion . A t o t a l of 30 ml of t h e a n t i s e r u m was c o l l e c t e d and p u r i f i e d a c c o r d i n g t o the p r o c e d u r e s d e s c r i b e d be l low . P u r i f i c a t i o n of Rabbi t A n t i s e r u m a g a i n s t t h e F a b Fragment  of MS 204 By u s i n g t h e P r o t e i n A a f f i n i t y c h r o m a t o g r a p h i c column, the mouse IgG f r a c t i o n s were o b t a i n e d f o l l o w i n g pH 2.2 e l u t i o n in 50 mM gy c i n e - H Q . In t o t a l about 15 mg of IgG was o b t a i n e d . S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s showed the p r e s e n c e of IgG h e a v y and l i g h t c h a i n s w i th p u r i t y g r e a t e r t h a n 90%. The c o l l e c t e d IgG f r a c t i o n s were t h e n l o a d e d onto an i r r e l e v a n t immunoaf f in i ty column. In t o t a l about 8 mg of IgG was washed out wi th PBS a t pH 8.0. The u n a d s o r b e d IgG f r a c t i o n s were t h e n l o a d e d onto MS 204 immunoaf f i n i t y column t o p u r i f y a n t i - F a b . A f t e r comple te washing w i th PBS, a peak - 46 -of O.D.280 was e l u t e d by G l y - H C l at pH 2.2. U l t i m a t e l y , 6 mg of A n t i - F a b IgG was o b t a i n e d from 30 ml of r a b b i t s e r u m . C h a r a c t e r i z a t i o n of Rabbi t A i d 204 I n d i r e c t i m m u n o f l u r e s c e n t a s s a y showed t h a t t h e F a b fragment of MS 204 c o u l d b ind t o t h e a c r o s o m a l r e g i o n of mouse sperm, which was i n h i b i t e d by the p r e s e n c e of p u r i f i e d A i d 204. The a n t i - F c a n t i b o d y d id not exhib i t any i n h i b i t i o n t o the b ind ing of e i t h e r MS 204 or the Fab fragment of MS 204 t o mouse sperm. A c t i v e Immunization wi th Aid 204 i n Mice When c o n j u g a t e d wi th hemocyanin and e m u l s i f i e d wi th F r u e n d ' s a d j u v a n t , A id 204 c o u l d e l i c i t an immune r e s p o n s e s t o Fab of A i d , as judged by E L I S A and double immunodi f fus ion . But t h e a p p e a r a n c e of a n t i - A i d 204 was only d e t e c t a b l e a f t e r the f i f t h immunizat ion (10 weeks). A f t e r the 7 th immunizat ion the a n t i s e r u m t i t e r was about 1:100,000 i n E L I S A wi th m i c r o p l a t e s c o a t e d wi th Fab of A id 204 ( n e g a t i v e c o n t r o l : 1:50). The s p e c i f i c r e a c t i o n of A id 204 t o a n t i - A i d 204 i s shown in F i g 8. The p r e s e n c e of an " i n t e r n a l image" of mouse sperm in Aid 204 was t e s t e d by i n d i r e c t e d i m m u n o f l u r e s c e n t a s s a y u s i n g m e t h a n o l - f i x e d mouse sperm. Under t h i s e x p e r i m e n t a l - 47 -c o n d i t i o n , a n t i - A i d 204 c o u l d b ind t o the a c r o s o m a l r e g i o n of mouse sperm. Compared wi th the p o s i t i v e c o n t r o l MS 204, A id 204 bound t o a s i m i l a r r e g i o n on the a c r o s o m e as o b s e r v e d under 1,000X f l u o r e s c e n t m i c r o s c o p e , but t h e b ind ing was weaker t h a n t h a t of MS 204 (F ig 9). A n t i f e r t i l i t y E f f e c t of A n t i - A i d 204 A n t i s e r a The r e s u l t s of in v i t r o f e r t i l i z a t i o n experiment u s i n g mouse sperms and mouse ova were summarized i n T a b l e IV. A p a r t i a l , but s i g n i f i c a n t i n h i b i t o r y e f f e c t on the mouse f e r t i l i z a t i o n was observed(41.0% v s . 82.8% i n c o n t r o l , p<0.05). The e f f e c t was much weaker t h a n wi th MS 204 (22.2%). - 48 -DISCUSSION G e n e r a t i o n of a n t i - i d i o t y p i c a n t i b o d i e s i s an a l t e r n a t i v e way t o o b t a i n a n t i g e n . As r e p o r t e d p r e v i o u s l y [ J e r n e , 1974; F a r i s and L o , 1985], a n t i - i d i o t y p i c a n t i b o d i e s c a n sometimes exh ib i t the r e c e p t o r b ind ing and p o s t - r e c e p t o r f u n c t i o n s of some hormones . F o r example, some a n t i b o d i e s a g a i n s t the i d i o t y p e of T S H a n t i b o d y c a n b ind t o T S H r e c e p t o r and e l i c i t a c t i o n s s i m i l a r t o t h o s e of T S H [Islam et a l , 1983]. T h i s a p p r o a c h would a l l o w us t o o b t a i n the "image" of c e r t a i n hormones or n e u r o t r a n s m i t t e r s which a r e d i f f i c u l t t o i s o l a t e f rom the b o d y . It i s e s p e c i a l l y u s e f u l i f the e p i t o p e of a t a r g e t a n t i g e n i s c o n f o r m a t i o n a l or i s s u g a r moie ty of a g l y c o p r o t e i n . As o b s e r v e d p r e v i o u s l y , t h e e p i t o p e r e a c t i v e wi th MS 204 may be c o n f o r m a t i o n a l . Its a n t i g e n i c i t y d i s a p p e a r s a f t e r homogeniz ing and d e t e r g e n t p r o c e s s i n g [unpubl i shed d a t a ] . But t h i s a n t i g e n seems t o be i m p o r t a n t in the mouse f e r t i l i z a t i o n p r o c e s s s i n c e i t b l o c k s mouse f e r t i l i z a t i o n a lmost c o m p l e t e l y in v i t r o . In t h i s s t u d y , the "image" of t h e e p i t o p e r e a c t i v e w i th MS 204 has b e e n c r e a t e in i t s a n t i - i d i o t y p e - - r a b b i t immunoglobul in. T h i s i s shown by 1), r a b b i t A id 204 i n h i b i t s t h e b i n d i n g of MS 204 t o mouse sperm; 2), a n t i - A i d 204 a n t i s e r u m c r o s s r e a c t s wi th mouse sperm; and - 49 -3), a n t i - A i d 204 a n t i s e r a i n h i b i t in v i t r o f e r t i l i z a t i o n of mouse ova by mouse sperm. T h i s i s t h e i n i t i a l s t e p t o w a r d s the i d e n t i f i c a t i o n and I s o l a t i o n of the fragment t h a t shows s i m i l a r or i d e n t i c a l a n t i g e n i c i t y of t h e o r i g i n a l a n t i g e n . A n a l y z i n g the s e q u e n c e of amino a c i d r e s i d u e s would enable us t o mass p r o d u c e t h e a n t i g e n i c fragment wi th chemica l or recombinant DNA methods . Immunoaf f i n i t y c h r o m a t o g r a p h y seems t o be a good method f o r the p u r i f i c a t i o n of a n t i - i d i o t y p i c a n t i b o d i e s f rom s e r u m . In t h i s s t u d y , a s e r i e s of immunoaf f i n i t y c h r o m a t o g r a p h i e s h a v e b e e n a p p l i e d . Among them, P r o t e i n A column does not seem t o be r e q u i r e d , but i t c a n i n c r e a s e t h e p u r i t y of IgG i n the o b t a i n e d s o l u t i o n . B e c a u s e the F c f ragments of mouse IgG a r e i d e n t i c a l , an i r r e l e v a n t mouse I g G - c o u p l e d S e p h a r o s e 4B c a n remove the a n t i b o d i e s a g a i n s t F c in the r a b b i t a n t i s e r u m . Then the a n t i - F a b of MS 204 was f i n a l l y o b t a i n e d f rom MS 2 0 4 - c o u p l e d immunoaf f i n i t y column. The o b t a i n e d Aid 204 ( r a b b i t IgG) i s weakly a n t i g e n i c in mouse as o b s e r v e d by the immunizat ion r e s u l t s . The a n t i - A i d 204 a n t i b o d i e s were d e t e c t e d only a f t e r 10 weeks of c o n s e c u t i v e immunizat ions . And the s t a i n i n g was r a t h e r weak i n i n d i r e c t i m m u n o f l u o r e s c e n t a s s a y as compared w i th MS 204 ( F i g . 9). F u r t h e r m o r e , as shown in T a b l e IV, the a n t i f e r t i l i t y e f f e c t of a n t i - A i d 204 a n t i b o d y i s much weaker t h a n i t s o r i g i n a l monoc lona l , MS 204. T h u s , i t s p o t e n t i a l - 50 -a p p l i c a t i o n in immunologica l f e r t i l i t y r e g u l a t i o n remains t o be i n v e s t i g a t e d . However , t h i s s t u d y h i g h l i g h t s a s t r a t e g y t o o b t a i n a c o n f o r m a t i o n a l e p i t o p e by means a n t i - i d i o t y p e s . - 51 -CONCLUSIONS 1. An a n t i - i d i o t y p i c a n t i b o d y , A i d 204, a g a i n s t monoc lona l s p e r m a n t i b o d y , MS 204, i s g e n e r a t e d by immunizat ion wi th MS 204 in r a b b i t , and i s p u r i f i e d by means of a f f i n i t y c h r o m a t o g r a p h y . The a n t i - A i d 204 a n t i s e r a c r o s s - r e a c t wi th mouse s p e r m a c r o s o m a l a n t i g e n which i s l o c a l i z e d in t h e same r e g i o n as t h a t r e a c t i v e wi th MS 204. T h i s impl ies t h a t the " i n t e r n a l image" of t h e s p e r m a n t i g e n e x i s t s in A id 204. 2. Immunization w i th A i d 204 i n mice p a r t i a l l y i n h i b i t s f e r t i l i z a t i o n . - 52 -APPENDIX. A SUMMARY ON GENERATION OF HYBRIDOMAS SECRETING MONOCLONAL ANTIBODIES OF IgA C L A S S AGAINST HUMAN SPERM As ment ioned in I n t r o d u c t i o n , monoc lona l a n t i b o d y of the IgA c l a s s a g a i n s t human s p e r m would p r o v i d e a good model t o s t u d y c l i n i c a l i n f e r t i l i t y [ C l a r k e , 1984; C l a r k e et a l , 1984; Naz et a l , 1985]. As p r e v i o u s l y r e p o r t e d , o r a l immunizat ion wi th a n t i g e n mixed wi th c h o l e r a t o x i n B and muramul d i p e p t i d e i s an e f f i c i e n t way t o s t i m u l a t e P e y e r ' s p a t c h and m e s e n t o r i c lymph nodes t o s e c r e t e a n t i b o d i e s of the IgA c l a s s [ E l s o n and E a l d i n g , 1984a,b; Taubman et a l , 1983]. B a s e d on t h i s b a c k g r o u n d , i n t a c t human sperm mixed wi th c h o l e r a t o x i n B and muramul d i p e p t i d e was u s e d t o o r a l l y immunize female B A L B / c mice. V e r y low a n t i b o d y t i t e r s were found in s e r u m and v a g i n a l washing . A f t e r f o u r c o n s e c u t i v e immunizat ions , the P e y e r ' s p a t c h e s and s p l e e n were t a k e n t o f u s e wi th N S - 1 myeloma c e l l s u s i n g t h e s e m i - s o l i d p h a s e method [Davis et a l , 1983]. Among 89 hybr idoma c o l o n i e s of P e y e r ' s p a t c h o r i g i n , and 226 of s p l e e n o r i g i n only one c e l l l i n e ( s p l een c e l l / N S - 1 ) s e r e t e d a n t i - s p e r m a n t i b o d y of IgG c l a s s . T h i s m o n o c l o n a l a n t i b o d y bound t o t h e p o s t - a c r o s o m e and t a i l of the methano l f i xed human sperm, but not t o the l i v e sperm. - 5 3 -In an a t t e m p t t o improve the e f f i c a c y of immunizat ion, I changed the immunizat ion reg imen. A female B A L B / c mouse was f i r s t pr imed by s .c . immunizat ion wi th homogenized human s p e r m e m u l s i f e d in F r e u n d ' s comple te a d j u v a n t , and t h e n b o o s t e d by o r a l immunizat ions . C e l l f u s i o n was c o n d u c t e d u s i n g P e y e r ' s p a t c h e s and s p l e e n c e l l s . But t h i s t ime P e y e r ' s p a t c h / N S - 1 hybr idomas f a i l e d t o grow. Among 126 s p l e e n / N S - 1 h y b r i d o m a s , 3 c o l o n i e s s e c r e t e d a n t i - s p e r m a n t i b o d i e s . Two of them were of IgG c l a s s , and one of IgM. Summarizing t h i s u n s u c c e s s f u l e f f o r t , I t h i n k t h a t s p e r m a n t i g e n s might be d e s t r o y e d in g a s t r o - i n t e n s t i n a l t r a c t , s o t h a t o r a l immunizat ion wi th sperm i s not an opt imal way t o s t i m u l a t e the g u t - a s s o i c a t e d immune s y s t e m . E f f o r t s s h o u l d be d i r e c t e d t o d i r e c t s t i m u l a t i o n t o P e y e r ' s p a t c h e s , i .e . , i n t r a - l y m p h node i n j e c t i o n . - 54 -T a b l e I. P u r i f i c a t i o n of sperm a n t i g e n , M S A - 2 0 7 , f rom t h e mouse t e s t i s . " A c t i v i t y " is d e f i n e d as the minimum O.D. v a l u e r e q u i r e d t o i n h i b i t t h e b ind ing of MS 207 t o mouse s p e r m in i n d i r e c t i m m u n o f l u o r e s c e n t i n h i b i t i o n a s s a y . SOLUBLE FRACTION 1st AFFINITY 2nd AFFINITY MOUSE TESTIS MOUSE TESTIS COLUMN COLUMN HOMOGENATE HOMOGENATE PURIFICATION PURIFICATION O.D./ML 81 28 0.183 0.051 VOLUME (ML) 20 27 13 8 TOTAL O.D. 1620 756 2.379 0.408 0.D./ACITVITY 16.2 5.6 0.037 0.006 FOLDS PURIFIED 1 2.9 438 2700 TOTAL ACTIVITY 324 151.2 63.7 63.5 YIELD (X) 100 46.7 19.7 19.6 PURITY (X) / / about 30 about 90 MOLECULAR HEIGHT / / / 600-700 (KDa) - 55 -T a b l e II. I n h i b i t o r y e f f e c t of monoc lona l a n t i b o d y , MS 207, and the c o r r e s p o n d i n g isoimmune s e r a r a i s e d a g a i n s t the p u r i f i e d mouse s p e r m a n t i g e n , M S A - 2 0 7 , on i n v i t r o f e r t i l i z a t i o n of mouse o v a . a: A n t i s e r a t o a d j u v a n t were u s e d as c o n t r o l . b: p<0.001 A N T I B O D I E S NO. NO. OVA NO. OVA F E R T I L I Z A T I O N ASSAYS EXAMINED F E R T I L I Z E D R A T E (%) C O N T R O L ( a ) 4 62 42 6 7 . 7 MS 207 3 76 6 7 . 9 ( b ) A N T I - M S A - 2 0 7 7 167 14 8 . 3 ( b ) - 5$ -T a b l e III. I n h i b i t o r y e f f e c t of isoimmune s e r a r a i s e d a g a i n s t p u r i f i e d s p e r m a c r o s o m a l a n t i g e n , M S A - 2 0 7 , on human s p e r m p e n e t r a t i o n t o z o n a - f r e e h a m s t e r o v a . a: a n t i s e r a t o a d j u v a n t were u s e d as c o n t r o l . b: p<0.001 A N T I B O D I E S NO. NO. OVA NO. OVA F E R T I L I Z A T I O N ASSAYS EXAMINED F E R T I L I Z E D R A T E (%) C O N T R O L ( a ) 3 45 28 6 2 . 2 A N T I - M S A - 2 0 7 4 97 4 4 . 1 ( b ) - 57 -TableIV. I n h i b i t o r y e f f e c t of mouse a n t i - r a b b i t A i d 204 and i t s c o r r e s p o n d i n g monoc lona l a n t i b o d y , MS 204, on in v i t r o f e r t i l i z a t i o n of mouse o v a . a: A n t i s e r a t o a d j u v a n t . b:p<0.01. c: p<0.001 A N T I B O D I E S NO. NO. OVA NO. OVA F E R T I L I Z A T I O N ASSAYS EXAMINED F E R T I L I Z E D R A T E (%) CONTROL 3 64 53 8 2 . 8 A N T I - A I D 204 3 39 16 4 1 . 0 MS 204 3 45 10 2 2 . 2 - 68 -F R A C T I O N NUMBER F i g 1. E l u t i o n p r o f i l e s f o r the p u r i f i c a t i o n of sperm a n t i g e n s r e a c t i v e t o MS 207 f rom mouse t e s t i s homogenate u s i n g an MS 207 immunoaf f i n i t y column. A: The p r o f i l e f o r t h e f i r s t p u r i f i c a t i o n . The e l u e n t f rom the f i r s t p u r i f i c a t i o n was f u r t h e r p u r i f i e d by t h e s e c o n d c h r o m a t o g r p h y , whose p r o f i l e i s shown under B. C and D d e n o t e t h e e l u t i o n w i th pH 10.0 and pH 11.5 b u f f e r , r e s p e c t i v e l y . - 59 -4 001-8 T E P S O F P U R I F I C A T I O N F i g 2. T h e y i e l d o f t o t a l a c t i v i t y o f M S A - 2 0 7 d u r i n g t h e p r o c e s s o f p u r i f i c a t i o n . 1: C r u d e h o m o g e n a t e o f m o u s e t e s t i s . 2: S o l u b l e f r a c t i o n o f t h e h o m o g e n a t e . 3: E l u e n t f r o m t h e f i r s t a f f i n i t y c h r o m a t o g r a p h y . 4: E l u e n t f r o m t h e s e c o n d a f f i n i t y c h r o m a t o g r a p h y . ( A c t i v i t y = t o t a l O.D./minimum O.D. e x h i b i t i n g i n h i b i o n o n t h e b i n d i n g o f MS 2 0 7 t o m o u s e s p e r m i n t h e i m m u n o f l u o r e s c e n t a s s a y . ) - 6 0 -ANTIBODY TITERS F i g 3. The r e s u l t s of e n z y m e - l i n k e d immunosorbent a s s a y (ELISA) showing t h e s p e c i f i c r e a c t i o n of MS 207 t o the p u r i f i e d mouse sperm a n t i g e n , MSA-207. The m i c r o t i t e r p l a t e was c o a t e d w i th MSA-207 (5 ug/ml). The a n t i b o d i e s were in s e r i a l s ^ j u t i o n . ,A:MS 207. B:an i r r e 1 e'vaitr^Hn onofel o n a 1 a n t i - s p e r m a n t i b o d y , HS 11. V - 61 -Fig 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 6% gel) showing the purity of MSA-207, a sperm antigen purified from mouse testis homogenate by an MS 207 immunoaff inity column. The molecular weight was shown to be 670 KDa. - 62 -u D MOLECULAR WEIGHT Ch 6 7 0 15 8 4 4 17 1.3 4-A \ \ B \ j . I I I _ i — i — 1 _ •i i i 1 1—1 H—1—1 l-H 1—1—-+-4— 10 13 16 19 TIME CHIN> F i g 5. The e l u t i o n p r o f i l e of M S A - 2 0 7 f rom G e l f i l t r a t i o n - h i g h p e r f o r m a n c e l i q u i d c h r o m t o g r a p h y (HPLC). A: t h e major peak w i th a n t i g e n i c a c t i v i t y . B: t h e peak of b u f f e r i m p u r i t y . - 63 -Fig 6. Indirect immunofluorescent assay showing that the isoimmune sera against MSA-207 crossreact with human sperm (1,000X). TIME C O U R S E CHEEKX F i g 7 . S e r u m a n t i b o d y t i t e r s f o l l o w i n g i m m u n i z a t i i o n s w i t h M S A - 2 0 7 i n f e m a l e m i c e . " I " d e n o t e s e a c h i m m u n i z a t i o n . T h e b a r s r e p r e s e n t s t a n d a r d d e v i a t i o n (SD) . - 65 -H " W W • 2. y 0 • ..! l._ \ • 5 " 7 0 A l-l T I B 0 D Y D I L. U T I 0 H F i g 8. T h e r e s u l t s o f e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) s h o w i n g t h e s p e c i f i c r e a c t i o n o f A i d 2 0 4 ( a n t i b o d y 2) t o t h e a n t i - A i d 2 0 4 ( a n t i b o d y 3) . T h e m i c r o t i t e r p l a t e w a s c o a t e d w i t h A: F a b f r a g m e n t o f A i d 2 0 4 , a n d B: a n i r r e l e v a n t m o n o c l o n a l a n t i b o d y , GO 1 5 . A n t i - A i d 2 0 4 w a s i n s e r i a l d i l u t i o n . - 66 -F i g 9. I n d i r e c t i m m u n o f l u o r e s c e n t a s s a y s h o w i n g t h a t h e t e r o i m m u n e s e r a a g a i n s t A i d 2 0 4 c r o s s r e a c t w i t h m o u s e s p e r m . T o p : t h e s t a i n i n g p a t t e r n o f MS 2 0 4 . B o t t o m : t h e s t a i n i n g p a t t e r n o f a n t i - A i d 2 0 4 . 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P.Sun, Y.Yang, A.C.Menge & C-Y.G.Lee: Purification and Characterization of a Sperm Antigen and Its Antifert i l i ty Effect When Actively Immunized, Abstract accepted to present on the Annual Meeting of the American Society of the Immunology of Reproduction, June 24 - 26, 1987, Indiana, USA. 4. C-Y. G. Lee, Y. Yang, P. Sun and F. Hu: Studies of Sperm Acrosomal Antigens Reactive to Monoclonal Antibodies that Inhibi Fertil ization, Abstract submitted to ASBC meeting. 5. C. 6. Lee, P. Sun and C. Kuo: Generation of Anti-idiotypic Antibodies to Monoclonal Sperm Antibodies and Evaluation of Their Antifert i l i ty Effects, Abstract submitted to the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, May 1-5, 1988, Las Vegas, Nevada, U.S.A. 6. M.Sc. Thesis at U.B.C: Studies of Sperm Antigens Using Specific Monoclonal Antibodies. Part One: Studies of a Sperm Acrosomal Antigen Reactive to a Monoclonal Antibody that Inhibits Ferti l izati ion Part Two: Generation, Purification and Characterization of an Anti-idiotypic Antibody that Crossreacts with a Sperm Conformational Antigen (Submitted to U.B.C.) 

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