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Studies of a sperm acrosomal antigen recognized by HS-63 monoclonal antibody Liu, Ming-Sun 1991

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STUDIES OF A SPERM ACROSOMAL ANTIGEN RECOGNIZED BY HS-63 MONOCLONAL ANTIBODY By Ming-Sun L i u B . S c , N a t i o n a l Chung-Hsing U n i v e r s i t y , Taiwan, 1981 M.Sc, N a t i o n a l Taiwan U n i v e r s i t y , Taiwan, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of O b s t e t r i c s and Gynaecology) (Reproductive and Developmental S c i e n c e s ) We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1991 © Ming-Sun L i u , 1991 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\)<>\(!\Y~:C5> cine] G>Wdecolo, The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT A sperm s p e c i f i c and species conserved monoclonal antibody (HS-63) was shown to i n h i b i t i n v i t r o f e r t i l i z a t i o n of mouse oocytes and human sperm penetration to zona-free hamster ova. The sperm antigen (SA-63) which reacts with HS-63 was found to be l o c a l i z e d on the sperm acrosome. Following sperm capacitation, t h i s antigen becomes exposed and i s shed aft e r the acrosome reaction. SA-63 may be involved i n the sperm acrosome reaction during the i n i t i a l f e r t i l i z a t i o n process. Sperm antigen (SA-63) from mouse (MSA-63) was p u r i f i e d from mouse testes with soluble and detergent extraction procedures followed by immunoaffinity chromatography. The p u r i f i e d MSA-63 antigen was shown to be a group of proteins with a size ranging from 25 Kd to 50 Kd and pis of about 4.2 when analyzed by two dimensional gel electrophoresis. MSA-63 antigen may be associated with actins i n i t s native form. A pr o t e o l y t i c a c t i v i t y was found in the solution of p u r i f i e d MSA-63 preparation. P u r i f i e d MSA-6 3 was used for immunization of mice and rabbits. Following successive immunizations, antisera of high i i t i t r e s were raised and reacted s p e c i f i c a l l y with sperm acre-some. The isoimmune sera from immunized mice exhibited s i g n i f i c a n t i n h i b i t i o n on i n v i t r o f e r t i l i z a t i o n of mouse oocytes. Complementary deoxyribonucleic acid (cDNA) fragments encoding the MSA-6 3 were cloned from a mouse t e s t i s cDNA l i b r a r y by using an immunoscreening method with rabbit antisera against MSA-63 as the detecting probe. When a s p e c i f i c cDNA probe was used for Northern blot analysis, an mRNA of 1.5 Kb i n size was detected only i n the adult mouse t e s t i s , but not i n any other somatic tissues. By Southern blot analysis, i t was also demonstrated that the gene encoding for SA-63 protein i s conserved among d i f f e r e n t mammalian species. The location of SA-63 antigen gene was determined to be on human chromosome 11 when analyzed with a blot of a human-hamster somatic c e l l hybrid panel. By DNA sequence analysis, a protein of 28 Kd i n size was deduced from the MSA-63 cDNA. The amino acid sequences of trypsin-digested peptide fragments of MSA-6 3 were used to v e r i f y that deduced amino acid sequence from the cDNA. The recombinant fusion proteins containing MSA-6 3 protein i i i fragment were produced i n EL. c o i i and used t o immunize female mice. S i m i l a r t o the o r i g i n a l HS-63 monoclonal antibody, the a n t i s e r a thus produced r e a c t e d o n l y with the sperm acrosome and r e v e a l e d s i g n i f i c a n t i n h i b i t i o n of the i n v i t r o f e r t i l i z a t i o n of mouse ooc y t e s . In the d e v e l o p i n g mouse t e s t i s , the e x p r e s s i o n o f MSA-63 gene was found t o be p o s t - m e i o t i c . P r o t e i n and mRNA of MSA-63 were not produced u n t i l day 20 a f t e r b i r t h . i v Table of Contents Page A b s t r a c t i i Table o f Contents v L i s t of Tables i x L i s t o f F i g u r e s x L i s t of A b b r e v i a t i o n s x i i Acknowledgments. x v i I. I n t r o d u c t i o n 1 A. Background and R a t i o n a l e 1 B. Formation of Sperm Acrosome 3 C. Role of Sperm Acrosome During F e r t i l i z a t i o n 4 D. T e s t i c u l a r S y n t h e s i s of Sperm P r o t e i n s i n Sperma togenes i s 8 E. A p p l i c a t i o n of Monoclonal Antibody i n Sperm Research... 9 F. Development of Sperm Antigen-Based Immunocontraceptive Vaccine 13 I I . M a t e r i a l s and Methods 18 A. Immunological techniques 18 1. Chemicals 18 2. Animals 18 3. P u r i f i c a t i o n o f Antibody from A s c i t e s F l u i d and A n t i s e r a 18 4. L a b e l i n g of Antibody 20 a. I o d i n a t i o n of Antibody 20 b. P r e p a r a t i o n of F I T C - l a b e l e d Antibody 21 c. P r e p a r a t i o n of Horse Radish Peroxidase-Labeled Antibody 21 5. I n d i r e c t Immunof l u o r e s c e n t Assay 22 a. P r e p a r a t i o n of Sperm S l i d e s 22 b. Immunof l u o r e s c e n t Assay 23 6. D i r e c t Immunof l u o r e s c e n t Assay 25 7. Enzyme-Linked Immunosorbent Assay (ELISA) 25 a. P r e p a r a t i o n of M i c r o t i t e r P l a t e s 26 b. Assay Procedures 26 8. Radioimmunosorbent Assay 27 9. P r o d u c t i o n of P o l y c l o n a l A n t i s e r a 27 B. P r o t e i n Chemistry 29 1. Chemicals 29 v 2. Determination of P r o t e i n C o n c e n t r a t i o n 29 3. D e t e c t i o n of Antigen Immunoactivity 30 4. P r e p a r a t i o n of Immunoaffinity Gel 30 5. P u r i f i c a t i o n of MSA-63 Antigens From Mouse T e s t e s . . 31 a. C onventional Method Combined wi t h Immunoaffinity Chromatography 31 b. Immunoaffinity Chromatography with Detergent E x t r a c t i o n 32 6. Gel F i l t r a t i o n 33 7. SDS P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s 34 a. Procedure of E l e c t r o p h o r e s i s . 34 b. P r o t e i n S t a i n i n g by Coomassie B r i l l i a n t Blue 35 c. P r o t e i n S t a i n i n g by S i l v e r Reagent 36 8. Two-Dimensional Gel E l e c t r o p h o r e s i s 36 9. Immunoblotting A n a l y s i s 37 a. Dot B l o t Assay 37 b. Western B l o t Assay 38 10. D e g l y c o s y l a t i o n of P r o t e i n 39 11. Amino A c i d Sequence A n a l y s i s 40 C. E v a l u a t i o n of Antibody on Sperm F u n c t i o n 41 1. Chemicals 41 2. Animals 41 3. In V i t r o F e r t i l i z a t i o n of Mouse Oocytes 42 a. I n d u c t i o n of S u p e r o v u l a t i o n 42 b. Sperm P r e p a r a t i o n 42 c. Sperm-Egg I n t e r a c t i o n 43 4. Sperm P e n e t r a t i o n to Zona-Free Hamster Ova 44 a. I n d u c t i o n of S u p e r o v u l a t i o n 44 b. P r e p a r a t i o n of Human Sperm 44 c. Sperm-Egg I n t e r a c t i o n 45 5. I n h i b i t i o n t o Acrosome R e a c t i o n 46 6. In V i v o Mating Experiments 47 7. S t a t i s t i c a l A n a l y s i s 47 D. M o l e c u l a r B i o l o g i c a l Methodology 48 1. M a t e r i a l s 48 2. B a c t e r i a S t r a i n 48 3. DNA I s o l a t i o n 48 a. T i s s u e DNA P r e p a r a t i o n 4 9 b. Bacteriophage DNA P r e p a r a t i o n 49 c. Plasmid DNA P r e p a r a t i o n 51 4. RNA I s o l a t i o n 53 5. Nick T r a n s l a t i o n of DNA 54 6. Southern B l o t H y b r i d i z a t i o n 55 a. Southern T r a n s f e r 55 b. H y b r i d i z a t i o n 55 7. Northern B l o t H y b r i d i z a t i o n . . . ' 56 8. Immunoscreening of Lambda g t 11 cDNA L i b r a r y . . . . . . . 56 a. A m p l i f i c a t i o n of cDNA L i b r a r y 56 b. P o l y c l o n a l A n t i s e r a P r e p a r a t i o n 57 c. Procedures of Immunoscreening 57 d. I d e n t i f i c a t i o n of P o s i t i v e Clones 61 9. Subcloning of DNA I n s e r t 61 a. P r e p a r a t i o n of Recombinant Plasmid DNA from v i Recombinant Bacteriophage 61 b. jEj. c o l i T r a nsformation 62 10. DNA sequencing 63 a. P r e p a r a t i o n and A l k a l i n e Denaturation of Plasmid DNA 63 b. Enzyme Rea c t i o n f o r Sequencing 64 c. Sequencing 65 11. P r o d u c t i o n of Recombinant F u s i o n P r o t e i n 65 a. Lysogenic S t r a i n P r e p a r a t i o n 65 b. P r e p a r a t i o n of Fusion P r o t e i n 66 12. Screening o f cDNA l i b r a r y by DNA probe 67 E. Immunohistochemistry 68 I I I . R e s u l t s 69 P a r t I . C h a r a c t e r i z a t i o n of HS-63 Monoclonal Antibody and E v a l u a t i o n of I t s Corresponding Sperm Antigens 69 A. L o c a t i o n of SA-63 Antigen i n Sperm 69 B. Epitope E v a l u a t i o n of HS-63 72 C. E v a l u a t i o n of HS-63 on Sperm F u n c t i o n 75 1. In V i t r o F e r t i l i z a t i o n of Mouse Oocytes 75 2. Human Sperm P e n e t r a t i o n Assay 79 3. The E f f e c t of HS-63 on Induced Acrosome R e a c t i o n . . . 81 4. In V i v o Mating Experiment by P a s s i v e Immunization i n Mouse ." 81 P a r t I I . 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 MSA-63 and E v a l u a t i o n of A n t i f e r t i l i t y E f f e c t s by I t s Isoimmune Sera 84 A. P u r i f i c a t i o n of HS-6 3 s p e c i f i c Antigens From Mouse Te s t e s (MSA-63) 84 1. Determination of S u b c e l l u l a r D i s t r i b u t i o n of MSA-63 84 2. P u r i f i c a t i o n of MSA-63 Antigen 85 B. C h a r a c t e r i z a t i o n of MSA-63 Antigens 90 1. A n a l y s i s of P u r i f i e d MSA-63 by Two-Dimensional SDS-gel E l e c t r o p h o r e s i s 90 2. Primary s t r u c t u r e A n a l y s i s of P r o t e i n Spots of MSA-63 Antigen on 2D-gels 96 3. M o l e c u l a r Weight A n a l y s i s of P u r i f i e d MSA-63 P r o t e i n 102 4. I n t e r a c t i o n A n a l y s i s Between MSA-63 and A c t i n 103 5. A n a l y s i s of P u r i f i e d MSA-63 P r o t e i n s by Enzymatic D e g l y c o s y l a t i o n 104 C. P r o d u c t i o n and E v a l u a t i o n of A n t i s e r a on F e r t i l i z a t i o n 108 v i i P a r t I I I . C l o n i n g of MSA-63 cDNA and Pr o d u c t i o n of I t s Fusion P r o t e i n . . . 115 A. Immunoscreening of Mouse T e s t i s cDNA L i b r a r y 116 B. Screening of the Mouse cDNA L i b r a r y by Using the C l o n i n g DNA As Probe 120 C. DNA Sequence A n a l y s i s 123 D. Pr o d u c t i o n and C h a r a c t e r i z a t i o n of MSA-63 F u s i o n P r o t e i n 133 1. P r e p a r a t i o n of Recombinant MSA-6 3 Fus i o n P r o t e i n from Lysogenic S t r a i n of E_s_ c o i i 133 2. I s o l a t i o n of Fusion P r o t e i n and E v a l u a t i o n of Corresponding A n t i s e r a 139 P a r t IV. Developmental S t u d i e s of MSA-63 Antigen 146 A. Conserved Nature of SA-63 Gene and I t s L o c a t i o n on Human Chromosome 146 B. T i s s u e - S p e c i f i c E x p r e s s i o n of MSA-63 Gene 151 C. E x p r e s s i o n of MSA-6 3 Gene i n Developing Mouse T e s t i s 151 D. Immunohistochemical S t u d i e s of MSA-63 Ant i g e n i n Mouse T e s t i s 156 IV. D i s c u s s i o n 161 Pa r t I . C h a r a c t e r i z a t i o n of HS-63 R e a c t i v e Sperm Antigens and E v a l u a t i o n of t h e i r Roles During F e r t i l i z a t i o n Processes 161 Pa r t I I . 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 MSA-63 170 P a r t I I I . C l o n i n g and C h a r a c t e r i z a t i o n of MSA-63 cDNA 175 Pa r t IV. Developmental S t u d i e s of MSA-6 3 A n t i g e n 180 V. C o n c l u s i o n and Future Research D i r e c t i o n 184 References 188 Appendix 202 v i i i L i s t o f Tables Page Table 1. I n h i b i t o r y e f f e c t of HS-63 monoclonal antibody on i n v i t r o f e r t i l i z a t i o n o f mouse oocytes 76 Table 2. I n h i b i t o r y e f f e c t of p u r i f i e d HS-63 on i n v i t r o f e r t i l i z a t i o n o f mouse oocytes..' 78 Table 3. I n h i b i t o r y e f f e c t of p u r i f i e d HS-63 on human sperm p e n e t r a t i o n of zona-free hamster ova 80 Table 4. E f f e c t s of a n t i b o d i e s on the i n v i v o f e r t i l i z a t i o n of mouse oocytes f o l l o w i n g p a s s i v e immunization and mating experiments 83 Table 5. P u r i f i c a t i o n of MSA-63 from mouse t e s t i s by two p u r i f i c a t i o n procedures 88 Table 6. Amino a c i d sequence a n a l y s i s of P u r i f i e d MSA-63.... 100 Table 7. I n h i b i t o r y e f f e c t of p o l y c l o n a l a n t i s e r a r a i s e d a g a i n s t MSA-63 on i n v i t r o f e r t i l i z a t i o n o f mouse oocytes... 114 Table 8. Amino a c i d composition of the deduced MSA-63 p r o t e i n 129 Table 9. I n h i b i t o r y e f f e c t of mouse a n t i s e r a r a i s e d a g a i n s t MSA-6 3 f u s i o n p r o t e i n on i n v i t r o f e r t i l i z a t i o n of mouse oocytes 145 i x L i s t of F i g u r e s Page F i g u r e 1. P r o t o c o l of immunoscreening of cDNA l i b r a r y w i t h antibody probe 59 F i g u r e 2. I n d i r e c t immunofluorescent s t a i n i n g of mouse and human sperm by u s i n g HS-63 monoclonal antibody . .... 70 F i g u r e 3. Immunofluorescent s t a i n i n g o f c a p a c i t a t e d mouse sperm by u s i n g F I T C - l a b e l e d HS-63 73 F i g u r e 4. F r a c t i o n a t i o n of the s o l u b l e supernatant of crude mouse t e s t e s homogenate by D E A E - c e l l u l o s e chromatography.... 86 Fi g u r e 5. ELISA showing the s p e c i f i c r e a c t i o n of HRP-labeled HS-63 monoclonal antibody t o the p u r i f i e d MSA-63 91 F i g u r e 6. Two-dimensional SDS-PAGE and Western b l o t a n a l y s i s of p u r i f i e d MSA-63 from mouse t e s t i s 93 F i g u r e 7 High-performance l i q u i d chromatography on r e v e r s e phase column of t r y p s i n - d i g e s t e d p u r i f i e d MSA-63 a n t i g e n . . . . 97 F i g u r e 8. Sephacryl S-300 g e l f i l t r a t i o n chromatography and SDS-PAGE to r e v e a l the molecular s i z e o f p u r i f i e d MSA-63 an t i g e n 104 F i g u r e 9. Sephacryl S-300 g e l f i l t r a t i o n chromatography t o r e v e a l the i n t e r a c t i o n between o L - a c t i n and p u r i f i e d MSA-63.. 106 F i g u r e 10. Enzymatic d e g l y c o s y l a t i o n o f MSA-63 p r o t e i n u s i n g N - g l y c o s i d a s e and O-g l y c o s i d a s e 109 Fi g u r e 11. ELISA showing the b i n d i n g between HS-63, mouse isoimmunesera, r a b b i t anti-MSA-63 s e r a or u n r e l a t e d a n t i b o d i e s and the p u r i f i e d MSA-63 coated on mi c r o w e l l s I l l F i g u r e 12. Immunoscreening of mouse t e s t i s cDNA l i b r a r y by u s i n g r a b b i t anti-MSA-63 s e r a as the d e t e c t i n g probe 116 Fi g u r e 13. Southern b l o t a n a l y s i s of c r o s s h y b r i d i z a t i o n of immuno-screened p o s i t i v e c l o n e s 118 Fi g u r e 14. Southern b l o t a n a l y s i s of p o s i t i v e MSA-63 cDNA cl o n e s 121 F i g u r e 15. R e s t r i c t i o n map and sequencing s t r a t e g y f o r the cDNA i n s e r t of MSA-63 124 Fi g u r e 16. N u c l e o t i d e sequence and deduced amino a c i d sequence of MSA-63 126 Fi g u r e 17. H y d r o p h i l i c i t y p l o t f o r the deduced MSA-63 amino a c i d sequence 131 x. F i g u r e 18. SDS-PAGE and Western b l o t assay o f recombinant MSA-63 f u s i o n p r o t e i n 134 F i g u r e 19. Percent i n h i b i t i o n of the b i n d i n g of 1 2 5 I - l a b e l e d HS-63 t o mi c r o w e l l s coated w i t h the s o l u b l e e x t r a c t of mouse sperm by the l y s a t e s of lysogens.... 137 Fi g u r e 20. I n d i r e c t immunofluorescent s t a i n i n g o f mouse sperm by u s i n g mouse a n t i s e r a a g a i n s t MSA-63 f u s i o n p r o t e i n . 140 F i g u r e 21. ELISA showing the b i n d i n g between mouse a n t i s e r a a g a i n s t MSA-63 f u s i o n p r o t e i n and mouse sperm homogenate coated on m i c r o w e l l s 142 F i g u r e 22. Southern b l o t a n a l y s i s of SA-63 gene from d i f f e r e n t mammalian s p e c i e s 147 F i g u r e 23. Southern b l o t a n a l y s i s of SA-63 gene by u s i n g the b l o t o f human-hamster somatic c e l l h y b r i d panel 149 F i g u r e 24. Northern b l o t a n a l y s i s of MSA-63 gene e x p r e s s i o n i n d i f f e r e n t mouse t i s s u e s 152 F i g u r e 25. Northern a n a l y s i s of RNA ob t a i n e d from mouse t e s t e s a t v a r i o u s stages o f development 154 F i g u r e 26. Ex p r e s s i o n o f MSA-63 an t i g e n i n dev e l o p i n g mouse t e s t e s 155 F i g u r e 27. Immunofluorescent assay of MSA-63 i n mouse t e s t e s a t v a r i o u s stages o f development 159 x i L i s t of A b b r e v i a t i o n s AR Acrosome Re a c t i o n BSA Bovine Sera Albumin BWW B i g g e r s , Whitten, and Whittingham medium cpm Counts Per Minute cDNA Complementary D e o x y r i b o n u c l e i c A c i d Con A Concanavalin A CTC C h l o r t e t r a c y c l i n e DEAE D i e t h y l a m i n o e t h y l DMSO Dimethyl s u l f o x i d e dNTP Deoxyribonucleoside t r i p h o s p h a t e DNA D e o x y r i b o n u c l e i c a c i d DNase Deoxyribonuclease DTT D i t h i o t h r e i t o l EDTA 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 ELISA Enzyme-linked Immunosorbent Assay FA-1 F e r t i l i z i n g - a s s o c i a t e d sperm a n t i g e n r e c o g n i z e d by monoclonal antibody MA-2 4 (Naz e t a l . , 1984a, 1986) FITC F l u o r e s c e i n - i s o t h i o c y a n a t e GA-1 A r a b b i t germ c e l l a n t i g e n i n v o l v e s i n f e r t i l i z a t i o n (Naz e t a l . , 1984b) HCG Human c h o r i o n i c gonadotrophin HPLC High Performance L i q u i d Chromatography HRP Horse Radish Peroxidase HS-21 A monoclonal antibody generated a g a i n s t human sperm (Wolf e t a l . , 1983, 1985) x i i HS-6 3 A monoclonal antibody generated a g a i n s t human sperm (Lee e t a l . , 1984a) IEF I s o e l e c t r i c F ocusing IgA Immunoglobulin A IgG Immunoglobulin G IMDM Iscove's M o d i f i e d Dulbecco's Media IPTG Iso p r o p y l - B e t a - D - T h i o g a l a c t o p y r a n o s i d e IVF In V i t r o F e r t i l i z a t i o n IU I n t e r n a t i o n a l U n i t Kb K i l o b a s e P a i r Kd K i l o d a l t o n LB L u r i a - B e r t a n i LDH-C4 L a c t a t e dehydrogenase-C4, a sperm s p e c i f i c isozyme of LDH (Goldberg, 1973) M29 A monoclonal antibody generated a g a i n s t mouse sperm ( S a l i n g e t a l . , 1986) M4 2 A monoclonal antibody generated a g a i n s t mouse sperm ( S a l i n g e t a l . , 1986) MA24 A monoclonal antibody generated a g a i n s t human sperm (Naz e t a l . , 1984a) ml M i l l i l i t e r mci M i l l i C u r i e s moi M u l t i p l i c i t y of I n f e c t i o n mRNA Messenger RNA MSA-63 Mouse sperm ant i g e n s p e c i f i c t o HS-63 monoclonal antibody (Lee e t a l . , 1984a) MS-204 One of the monoclonal a n t i b o d i e s generated a g a i n s t mouse sperm a n t i g e n (Lee e t a l . , x i i i 1984b, 1986b) One of the monoclonal a n t i b o d i e s generated a g a i n s t mouse sperm a n t i g e n (Lee e t a l . , 1984b, 1986b) O p t i c a l D e n s i t y Phosphate B u f f e r e d S a l i n e P o l y e t h y l e n e G l y c o l 3-phosphoglycerate kinase-2 Plaque Forming U n i t A guinea p i g sperm a n t i g e n d e f i n e d by Primakoff (Primakoff e t a l . , 1988b) I s o e l e c t r i c P o i n t P h e n y l m e t h y l s u l f o n y l f l u o r i d e Pregnant mare's serum gonadotrophin Peanut a g g l u t i n i n . Pisum sativum l e c t i n P o l y v i n y l p y r r o l i d o n e (M.W. 40,000) R i c i n i s communis a g g l u t i n i n - I I Radioimmunoassay Radioimmunosorbent assay R i b o n u c l e i c a c i d Ribonuclease Rabbit sperm acrosomal a n t i g e n d e f i n e d by O'Rand (O'Rand e t a l . , 1984) . Sperm a n t i g e n of v a r i o u s s p e c i e s c r o s s -r e a c t i v e t o HS-63 monoclonal antiboby ( L i u e t a l . , 1990) Sodium Dodecyl S u l f a t e x iv SP-10 Human sperm acrosomal a n t i g e n 10.defined by Herr (Herr e t a l . , 1990a) SPA Sperm P e n e t r a t i o n Assay PAGE Polya c r y l a m i d e Gel E l e c t r o p h o r e s i s TEMED N,N,N',N'-Tetramethylethylenediamine TFA T r i f l o u r o a c e t i c a c i d T r i s Tri(hydroxymethyl)aminomethane tRNA T r a n s f e r RNA u l M i c r o l i t e r X-Gal 5-Bromo-4-Chloro-3-Indoyl-Beta-D-Ga l a c t o p y r a n o s i d e ZP-3 Zona p e l l u c i d a e p r o t e i n - 3 w i t h molecular weight of about 8.5-9 Kd on SDS-gel xy Acknowledgments I would l i k e t o thank a l l members who helped me t o complete t h i s t h e s i s work. F i r s t o f a l l , I am ve r y g r a t e f u l t o my s u p e r v i s o r , Dr. Gregory Lee. During t h i s p e r i o d , he gave me f u l l support i n my r e s e a r c h work e s p e c i a l l y p r o v i d i n g me o p p o r t u n i t i e s to l e a r n many techniques from other l a b s . I would l i k e t o g i v e my a p p r e c i a t i o n t o Dr. C h r i s Lau a t U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o f o r h i s i n s t r u c t i o n i n cDNA c l o n i n g , Dr. Ruedi A e b e r s o l d a t Biomedical Research Center of B r i t i s h Columbia f o r h i s i n s t r u c t i o n i n p r o t e i n sequence a n a l y s i s and Dr. Mi c h a e l Hayden a t Department of M e d i c a l G e n e t i c s f o r the p e r m i s s i o n t o use the f a c i l i t i e s of h i s l a b . I a l s o a p p r e c i a t e the advic e of Dr. Angus Tseng on my t h e s i s work. Furthermore, I would l i k e t o thank my f a m i l y f o r s u p p o r t i n g my s t u d i e s . F i n a l l y , I am ple a s e d t o t e l l my wif e " Now, we can have the t h i r d k i d . " x v i I. INTRODUCTION AJ:_ HISTORICAL BACKGROUND AND RATIONALE During the l a s t decade, a number of monoclonal sperm a n t i b o d i e s have been generated i n our l a b o r a t o r y (Lee e t a l , 1984a,b,c). The i n h i b i t o r y e f f e c t s of these a n t i b o d i e s on sperm f u n c t i o n and f e r t i l i z a t i o n have a l s o been assessed (Lee e t a l , 1987; Menge e t a l , 1987). Among these a n t i b o d i e s , HS-63 was shown to r e a c t with an acrosomal a n t i g e n o f sperm from s e v e r a l mammalian s p e c i e s i n c l u d i n g human, r a b b i t , and mouse, but d i d not r e a c t with any somatic t i s s u e s (Anderson e t a l . , 1987). T h i s h i g h l y s p e r m - s p e c i f i c and s p e c i e s - c o n s e r v e d antibody has been demonstrated t o i n h i b i t both i n v i v o and i n v i t r o f e r t i l i z a t i o n of mouse (Lee e t a l , 1986b), and human sperm p e n e t r a t i o n of zona-f r e e hamster ova (Menge e t a l , 1987). A c c o r d i n g t o the r e p o r t of i n t e r - l a b o r a t o r y e v a l u a t i o n s sponsored by the World H e a l t h O r g a n i z a t i o n i n 1986, HS-63 has been recommended wit h h i g h p r i o r i t y f o r the f u t u r e development of immunocontraceptive v a c c i n e s (Anderson e t a l . , 1987). 1 Judging from the background i n f o r m a t i o n , i n the f i r s t p a r t of my t h e s i s , the HS-63 monoclonal antibody was c h a r a c t e r i z e d and used as a probe t o i n v e s t i g a t e the l o c a t i o n and p o s s i b l e p h y s i o l o g i c a l f u n c t i o n of the corres p o n d i n g sperm a n t i g e n , SA-63, i n the processes of f e r t i l i z a t i o n . In the second p a r t , d e t a i l e d procedures of 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 o f the mouse sperm a n t i g e n (MSA-63) were worked out. In a d d i t i o n , the a n t i f e r t i l i t y e f f e c t s of a c t i v e and p a s s i v e immunizations of MSA-63 i n mice were a l s o e v a l u a t e d . These data are c o n s i d e r e d t o be e s s e n t i a l f o r the f u t u r e development of sperm antigen-based immunocontraceptive v a c c i n e s . In the t h i r d p a r t , c l o n i n g of cDNA fragments encoding the MSA-6 3 and i t s c h a r a c t e r i z a t i o n were c a r r i e d out. The recombinant f u s i o n p r o t e i n s c a r r y i n g MSA-63 p r o t e i n fragments produced by such techniques were used t o generate a n t i s e r a f o r the assessment of a n t i f e r t i l i t y e f f e c t . In the f o u r t h p a r t , the corresponding HS-63 monoclonal antibody, p o l y c l o n a l a n t i s e r a and cDNA probes were used as probes f o r developmental s t u d i e s of SA-63 a n t i g e n s y n t h e s i s d u r i n g 2 spermatogenesis. Bj. FORMATION OF SPERM ACROSOME. The sperm acrosome i s a membrane-bound, ly s o s o m e - l i k e o r g a n e l l e l o c a l i z e d i n the a n t e r i o r r e g i o n of sperm head, j u s t above the nucleus and beneath the plasma membrane (Mann, 1964, P h i l i p s , 1972). Between the o u t e r and i n n e r acrosomal membranes, a v a r i e t y of h y d r o l y t i c enzymes i n c l u d i n g p r o t e i n a s e s , g l y c o s i d a s e s , phosphatases, a r y l s u f a t a s e s , and phospholipase are pre s e n t i n the acrosome. Some of these enzymes may be i n t e g r a l or a s s o c i a t e d components of the acrosomal membranes (Hartmann, 1983). The acrosome f i r s t appears as a product of the G o l g i complex i n spermatids d u r i n g spermiogenesis (Fawcett and Bedford, 1979). A c c o r d i n g to Leblond and Clermont (1952), spermiogenesis of r a t s i s s u b d i v i d e d i n t o 19 stages of development based on the changes of the acrosomic system and nucleus of the spermatid. In stage 2 of spermiogenesis, the h e m i s p h e r i c a l G o l g i apparatus c o n t a i n s s e v e r a l proacrosomic g r a n u l e s i n i t s medulla. These granules fuse i n t o a s i n g l e acrosomic granule which migrates towards the 3 nucleus and becomes a t t a c h e d t o i t while f u r t h e r m a t e r i a l i s added from the G o l g i apparatus. The acrosome then spreads over p a r t of the n u c l e a r s u r f a c e . ROLE OF SPERM ACROSOME DURING FERTILIZATION When r e l e a s e d from the t e s t i s , spermatozoa are not competent t o f e r t i l i z e eggs ( A u s t i n and Short, 1982). They must undergo a process of maturation d u r i n g the t r a n s i t i n the epididymis t o ga i n t h e i r p r o g r e s s i v e m o t i l i t y (Hamilton, 1977, Hoskins e t a l . , 1973). Besides the epididymal maturation, mammalian sperm r e q u i r e an the c a p a c i t a t i o n process which occurs w i t h i n the female r e p r o d u c t i v e t r a c t , t o prepare, them f o r the acrosome r e a c t i o n and f e r t i l i z a t i o n (Chang, 1951; A u s t i n , 1951; Yanagimachi, 1981; Mcore and Bedford, 1983). A number of changes of sperm have been r e p o r t e d t o r e l a t e t o sperm c a p a c i t a t i o n (Ahuja, 1985). These changes i n c l u d e i n c r e a s e o f m o t i l i t y , rearrangements of intramembranous p a r t i c l e s , removal of sperm s u r f a c e components and a decrease i n net s u r f a c e n egative charge (Longo, 1987). 4 A f t e r the c a p a c i t a t i o n process, a spermatozoon wi t h an i n t a c t acrosome cannot p e n e t r a t e an egg. The acrosome must ' r e a c t ' o r break down, t o r e l e a s e i t s contents p r i o r t o sperm p e n e t r a t i o n . The s u c c e s s i v e stages of t h i s event are g e n e r a l l y c a l l e d the acrosome r e a c t i o n (AR) ( A u s t i n and Short, 1982) . I t i s g e n e r a l l y b e l i e v e d t h a t the acrosome undergoes changes when the spermatozoon comes i n t o c o n t a c t with the egg (Dan, 1967? Clowin and Clowin, 1967). In mammals, the plasma membrane o v e r l a y i n g the acrosome and the out e r acrosomal membrane, fuse a t m u l t i p l e s i t e s and form an a r r a y of v e s i c l e s d u r i n g the acrosome r e a c t i o n (Barros e t a l . , 1967). The v e s i c l e s thus formed have been shown t o be mosaics c o n s i s t i n g of membranes d e r i v e d from both the plasma and the outer acrosomal membrane ( R u s s e l l e t a l . , 1979). At the l e v e l of the e q u a t o r i a l segment, the plasma membrane and the acrosomal membrane are fused t o maintain a continuous membrane t h a t d e l i m i t s the contents of the spermatozoon. As a r e s u l t o f the acrosome r e a c t i o n , the v e s i c l e s are d e r i v e d from the f u s i o n of plasma and out e r acrosomal membranes, 5 and subsequently the contents of the acrosome are r e l e a s e d t o the surrounding environment. T h e r e f o r e , the r e l e a s e of acrosomal contents i s c o n s i d e r e d t o be an e x o c y t o t i c event. About a dozen of the enzymes known t o e x i s t i n the acrosome are r e l e a s e d d u r i n g the acrosome r e a c t i o n (Mack e t a l . , 1983). Apparently, the f u n c t i o n of these enzymes i s t o a s s i s t the sperm t o pen e t r a t e the egg. I n h i b i t o r s and a n t i b o d i e s t o these sperm acrosomal enzymes are known t o b l o c k f e r t i l i z a t i o n a t the l e v e l of the zona p e l l u c i d a (Dunbar e t al.,1976; P e r r e a u l t e t a l . , 1979). The t i m i n g of the acrosome r e a c t i o n i n the normal course of mammalian f e r t i l i z a t i o n i s c o n t r o v e r s i a l (Moore and Bedford, 1983). I t i s g e n e r a l l y b e l i e v e d t h a t sperm should complete the acrosome r e a c t i o n p r i o r t o p e n e t r a t i o n through the zona p e l l u c i d a . The most l i k e l y s i t e s f o r t h i s r e a c t i o n are w i t h i n the cumulus and a t the s u r f a c e of the zona p e l l u c i d a (Florman and Storey, 1982; S a l i n g and Storey, 1979). A number of s t u d i e s i n d i c a t e d t h a t sperm should f i r s t p e netrate the i n t a c t cumulus of the oocyte f o l l o w e d by b i n d i n g t o the zona p e l l u c i d a and then undergo the acrosome r e a c t i o n (Florman and Storey, 1982; Storey e t a l . , 1984). C a p a c i t a t e d hamster sperm wi t h i n t a c t acrosome can b i n d t o the zona p e l l u c i d a whereas sperm which have undergone the 6 acrosome r e a c t i o n f a i l t o do so. Furthermore, zona p r o t e i n s which r e c o g n i z e mouse sperm plasma membrane r e c e p t o r s have been i d e n t i f i e d from the zona p e l l u c i d a of mouse eggs ( B l e i l and Wassarman, 1980a,b, 1983). One of the zona p r o t e i n s , ZP-3, has a l s o been proven t o induce the acrosome r e a c t i o n i n v i t r o ( B l e i l and Wassarman, 1983, 1986; Wassarman, 1987a,b). However, r e c e n t s t u d i e s i n d i c a t e t h a t the sperm acrosome r e a c t i o n may not be induced by zona p e l l u c i d a o n l y ( B a v i s t e r , 1982; Lenz e t a l . , 1982; Reyes e t a l . , 1984; S i i t e r i e t a l . , 1988; T e s a r i k , 1985). In view of the complicated processes i n v o l v e d i n the sperm acrosome r e a c t i o n , s e v e r a l techniques have been developed t o analyze t h i s event (Soupart and Strong, 1974; McMaster e t a l . , 1978; S a l i n g and Storey, 1979; T a l b o t and Chacon, 1981; Wolf e t a l . , 1985). Methods are now a v a i l a b l e t o d i f f e r e n t i a t e acrosome-i n t a c t from acrosome-reacted sperm a t the l i g h t m i c r o s c o p i c l e v e l S e v e r a l f l u o r e s c e i n 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 l e c t i n s are wi d e l y used t o demonstrate the presence or absence of sperm acrosome d u r i n g the acrosome r e a c t i o n . These i n c l u d e d R i c i n i s communis a g g l u t i n i n - I I (RCA-II) ( T a l b o t and Chacon, 1980), .Pisum sativum a g g l u t i n i n (PSA) (Cross e t a l . , 1986), peanut a g g l u t i n i n 7 (PNA) (Takamune, 1987), and c o n c a n a v a l i n A (Con A) (Holden e t a l . , 1990). Although methodologies and t o o l s are a v a i l a b l e t o study the sperm acrosome r e a c t i o n , l i t t l e i s known about the d e t a i l e d m olecular events a s s o c i a t e d w i t h i s p r o c e s s . T h i s i s simply because of the l a c k of b i o c h e m i c a l markers f o r such s t u d i e s . P_i. TESTICULAR SYNTHESIS OF SPERM PROTEINS IN SPERMATOGENESIS G e n e r a l l y , genes expressed d u r i n g spermatogenesis can be c l a s s i f i e d i n t o t h r e e major c a t e g o r i e s : (a) genes t h a t are expressed e x c l u s i v e l y d u r i n g spermatogenesis, (b) genes encoding p r o t e i n s f o r which t h e r e i s s w i t c h i n g of i s o t o p e s or isozymes , (c) genes whose e x p r e s s i o n i s somehow a l t e r e d , e i t h e r q u a n t i t a t i v e l y or q u a l i t a t i v e l y , d u r i n g d i f f e r e n t i a t i o n ( W i l l i s o n and Ashworth, 1987). P o s t m e i o t i c gene e x p r e s s i o n d u r i n g spermatogenesis has been demonstrated by s e v e r a l known b i o c h e m i c a l markers such as 3-phosphoglycerate kinase-2 (PGK-2) ( E r i c k s o n e t a l . , 1980), protamine (Kleene e t a l . , 1984), o£-tubulin ( D i s t e l e t al.,1984; 8 Hecht e t a l . , 1984) and v a r i o u s u n i d e n t i f i e d mRNAs i n spermatids (Fujimoto and E r i c k s o n , 1982). Since acrosome formation i s i n i t i a t e d immediately a f t e r the completion of m e i o s i s , acrosomal p r o t e i n s c o u l d be s y n t h e s i z e d e i t h e r i n the p r e - or p o s t - m e i o t i c stage. R e c e n t l y , a number of acrosomal p r o t e i n s were found t o appear f i r s t i n the p o s t m e i o t i c stages (e.g. spermatids) of spermatogenesis (Lee and Wong, 1986a). I t remains to be determined whether the p o s t m e i o t i c e x p r e s s i o n takes p l a c e a t t r a n s c r i p t i o n a l or t r a n s l a t i o n a l l e v e l s , or i s simply due to the p o s t -t r a n s l a t i o n a l m o d i f i c a t i o n s of e a r l i e r expressed sperm p r o t e i n s (Kleene e t a l . , 1984). To c l a r i f y t h i s p o i n t , i t i s e s s e n t i a l t o have b i o c h e m i c a l markers which are a s s o c i a t e d with acrosome formation or the acrosome r e a c t i o n . G^ . APPLICATION OF MONOCLONAL ANTIBODIES IN SPERM RESEARCH Mammalian spermatogenesis i s an event i n which numerous bi o c h e m i c a l markers are turned on and o f f t o f u l f i l l s t r u c t u r a l , f u n c t i o n a l and developmental requirements of spermatozoa. P r i o r 9 t o sperm p e n e t r a t i o n and sperm-egg membrane f u s i o n , spermatozoa have t o go through maturation i n the e p i d i d y m i s , so t h a t they g a i n m o t i l i t y , the a b i l i t y t o undergo c a p a c i t a t i o n and the acrosome r e a c t i o n (Bedford and M i l l a r , 1979; Feuchter e t a l . , 1981) . To analyze such a complicated event, i t i s e s s e n t i a l t o have s p e c i f i c a n t i b o d i e s t h a t can r e a c t w i t h d i f f e r e n t a n t i g e n i c components of spermatozoa. Since the advent of hybridoma technology, i t has become p o s s i b l e t o generate monoclonal a n t i b o d i e s t h a t r e a c t with v a r i o u s a n t i g e n i c determinants of sperm s u r f a c e antigens (Lee e t a l . , 1984a,b; Lee and Wong, 1986a). These monoclonal a n t i b o d i e s can be v a l u a b l e t o o l s t o f o l l o w the development and the l o c a t i o n of p a r t i c u l a r sperm antigens i n the germ c e l l s a t d i f f e r e n t stages of spermatogenesis. The g e n e r a t i o n and c h a r a c t e r i z a t i o n of monoclonal a n t i b o d i e s a g a i n s t spermatozoa from s e v e r a l mammalian s p e c i e s were r e p o r t e d by many i n v e s t i g a t o r s . They were from guinea p i g s (Primakoff e t a l . , 1988a; Myles, 1987), from r a b b i t s (O'Rand and Irons, 1984; Lee e t a l . , 1984c), from mice ( S a l i n g e t a l . , 1983; O'Brien e t a l . , 1 9 8 8 ; Lee e t a l . , 1934b), from hamsters (Moore and Hartman, 10 1984), and from humans (Naz e t a l . , 1984a; Wolf e t a l . , 1983; Lee et a l . , 1984a; Herr e t a l . , 1990a). Monoclonal antibody HS-21 which was generated a g a i n s t human spermatozoa r e c o g n i z e d t a r g e t antigens r e s t r i c t e d t o the acrosomal cap determined by i n d i r e c t immunofluorescent assay (Wolf e t a l , 1985). I t was used as a marker f o r the e v a l u a t i o n of the acrosomal s t a t u s of spermatozoa. Monoclonal antibody 1D4 r e a c t s with a g l y c o c o n j u g a t e a n t i g e n of the d e v e l o p i n g mouse spermatid acrosome u n t i l the t e r m i n a l s t e p s of spermiogenesis (Gerton e t a l . , 1988). I t can not b i n d to the acrosome of mouse epididymal spermatozoa. However, 1D4 r e a c t s with the acrosomal r e g i o n of guinea p i g epididymal sperm. From the immmunoblots of e x t r a c t s of guinea p i g spermatocytes, round spermatids, condensing spermatids and sperm wi t h t h i s monoclonal antibody, the c o r r e s p o n d i n g a n t i g e n changes d u r i n g germ c e l l d i f f e r e n t i a t i o n can be demonstrated. Thus 1D4 can be used as a marker f o r s t u d i e s of the s y n t h e s i s , s t r u c t u r e , and assembly of sperm o r g a n e l l e s , such as the acrosome. Monoclonal antibody M42, which r e a c t s with a sperm acrosome 11 a n t i g e n o f 220 KD, was shown t o i n h i b i t f e r t i l i z a t i o n i n mice i n a dose-dependent manner ( S a l i n g e t a l . , 1986). T h i s antibody s p e c i f i c a l l y i n h i b i t s p h y s i o l o g i c a l l y zona-induced (ZP-3), but not p h a r m a c o l o g i c a l l y ionophore (A23187)-induced acrosome r e a c t i o n i n mouse sperm. When the f l u o r e s c e n t a n t i b i o t i c c h l o r t e t r a c y c l i n e (CTC) was used t o v i s u a l i z e the t h r e e d i f f e r e n t p a t t e r n s d u r i n g the acrosome r e a c t i o n (B, S, and AR), M42 was shown t o a r r e s t the zona-induced sperm acrosome r e a c t i o n i n the B-p a t t e r n . T h e r e f o r e , i t was concluded t h a t M42 b l o c k s the e a r l y s t e p i n the acrosome r e a c t i o n cascade and i s unable t o prevent subsequent events of the cascade once they have been i n i t i a t e d (Leyton e t a l . , 1989). Monoclonal sperm a n t i b o d i e s were a l s o used as probes t o analyze the developmental e x p r e s s i o n s of r e l e v a n t sperm s u r f a c e a n t i g e n s d u r i n g d i f f e r e n t stages of spermatogenesis (Lee and Wong, 1986a). By immunofluorescent assay, monoclonal sperm a n t i b o d i e s were used as probes t o s t a i n f r e s h l y i s o l a t e d t e s t i c u l a r c e l l s and t i s s u e s e c t i o n s from mouse t e s t e s of d i f f e r e n t ages. I t was g e n e r a l l y observed t h a t a s i g n i f i c a n t number of these a n t i b o d i e s r e a c t e d with c y t o p l a s m i c components of the spermatogenic c e l l s i n the t e s t i s a t the p o s t m e i o t i c stages 12 (e.g. day 22 a f t e r b i r t h ) . A f t e r m e i o s i s , the percentage o f se m i n i f e r o u s t u b u l e s s t a i n e d by i n d i r e c t immunofluorescence was found t o i n c r e a s e w i t h the age of mice. T h e r e f o r e , i t was suggested t h a t some c y t o p l a s m i c components ( e s p e c i a l l y acrosomal) of spermatogenic c e l l s are expressed p o s t m e i o t i c a l l y i n the t e s t i s and l a t e r t r a n s l o c a t e d t o the sperm s u r f a c e d u r i n g l a t e stages o f spermiogenesis (Lee and Wong, 1986a). The 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 these c o r r e s p o n d i n g sperm antigens are e s s e n t i a l steps f o r the e v a l u a t i o n of 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 the f e r t i l i z a t i o n p r o c e s s e s . However, very few sperm an t i g e n s have been p u r i f i e d and c h a r a c t e r i z e d . The main d i f f i c u l t i e s f o r such s t u d i e s a r i s e from the f a c t t h a t the m a j o r i t y o f these sperm s u r f a c e antigens are membrane-associated or i n t e g r a l membrane p r o t e i n s (Primakoff and Myles, 1983). F\ DEVELOPMENT OF SPERM ANTIGEN-BASED IMMUNOCONTRACEPTIVE  VACCINE Isoimmunization with sperm or t e s t i s e x t r a c t causes 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 i n animals (Menge e t a l . , 1979). Th e r e f o r e , the development of sperm antigen-based v a c c i n e s r e p r e s e n t s a promising approach to c o n t r a c e p t i o n (Talwar, 1986; Anderson e t a l . , 1987). However, crude e x t r a c t s of sperm or t e s t i s are not a p p r o p r i a t e t o be used d i r e c t l y as a v a c c i n e due t o the presence of numerous components which may c r o s s - r e a c t w i t h somatic t i s s u e s , c a u s i n g u n d e s i r a b l e immunopathological consequences (Mathur e t a l , 1981; Schachuer e t a l , 1975). T h e r e f o r e , i t i s e s s e n t i a l t o i d e n t i f y s p e r m - s p e c i f i c auto- and i s o - a n t i g e n s which may p r o p e r l y serve as e f f e c t i v e c o n t r a c e p t i v e v a c c i n e s . I t has been known f o r decades t h a t c e r t a i n sperm s u r f a c e components are auto- or iso-immunogenic (Tung, 1980; Menge e t a l , 1979). In view of the known e f f e c t s of sperm a n t i b o d i e s on sperm f u n c t i o n s and t h e i r a s s o c i a t i o n w i t h human i n f e r t i l i t y , sperm antigen-based c o n t r a c e p t i v e v a c c i n e s appear t o be an i d e a l c h o i c e f o r p r e - f e r t i l i z a t i o n c o n t r a c e p t i o n i n humans and animals (Anderson and Alexander, 1983). From c l i n i c a l o b s e r v a t i o n s , the i n d i v i d u a l s who s u f f e r from sperm a n t i b o d y - r e l a t e d i n f e r t i l i t y do not e x h i b i t .other h e a l t h problems compared t o the normal s u b j e c t s (Mathur e t a l , 1981; Schachuer e t a l , 1975). Since the c o n t r a c e p t i v e e f f e c t i s a s s o c i a t e d with the t i t r e s of c i r c u l a t i n g and/or 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 , the r e v e r s i b i l i t y of 14 t h i s c o n t r a c e p t i v e method has been r e p o r t e d i n animal s t u d i e s (Primakoff, 1988b). D e s i r a b l e sperm antigens c o u l d be i d e n t i f i e d and ob t a i n e d f o r c o n t r a c e p t i v e v a c c i n e developments from s e v e r a l s ources. These i n c l u d e (a) sperm antigens which 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 n t i b o d i e s from s e r a of i n f e r t i l e p a t i e n t s (Jackson e t a l , 1975; Teuscher e t a l , 1982 and 1983), (b) sperm p r o t e i n s o f known s p e r m - s p e c i f i c enzymes such as l a c t a t e dehydrogenase-C4 (LDH-C4) (Goldberg, 1973) and (c) sperm s u r f a c e antigens r e a c t i v e t o monoclonal a n t i b o d i e s which a f f e c t normal sperm f u n c t i o n s (Naz e t a l , 1984b and 1986; O'Rand e t a l , 1984; S a i l i n g e t a l , 1986; Primakoff e t a l , 1988a). Using hybridoma techniques, monoclonal a n t i b o d i e s , each o f which r e a c t s with a s i n g l e determinant of a g i v e n sperm a n t i g e n , can be generated. These monoclonal a n t i b o d i e s were used t o i n v e s t i g a t e the developmental and f u n c t i o n a l aspects of complementary sperm antigens (Lee e t a l , 1984a,b,c; Lee and Wong, 1986b; and Lee e t a l , 1987). Some monoclonal a n t i b o d i e s which r e a c t mainly with the sperm acrosome were found t o i n h i b i t the f e r t i l i z a t i o n of mouse oocytes i n v i t r o and i n v i v o and 15 i m p l a n t a t i o n of embryos (Lee e t a l , 1986a). T h e r e f o r e , the corresponding sperm a n t i g e n which r e a c t s with each of these a n t i b o d i e s may be a s u i t a b l e c h o i c e f o r the development of immunocontraceptives, i f the a n t i g e n s are i d e n t i f i e d , p u r i f i e d and c h a r a c t e r i z e d . Some of s p e r m - s p e c i f i c antigens which r e a c t with p o l y c l o n a l or monoclonal a n t i b o d i e s have been p u r i f i e d and e v a l u a t e d f o r t h e i r a n t i f e r t i l i t y e f f e c t s . L a c t a t e dehydrogenase-C4 i s a sperm-s p e c i f i c enzyme which mainly attaches t o the sperm t a i l . A c t i v e immunization of t h i s s p e r m - s p e c i f i c a n t i g e n was shown t o p a r t i a l l y suppress f e r t i l i t y i n experimental animals (Goldberg, 1973 and 1975; K i l l e and Goldberg, 1979 and 1980; Goldberg e t a l , 1981). Menge and h i s coworkers used a monoclonal sperm antibody to p u r i f y and c h a r a c t e r i z e GA-1, a germ c e l l a n t i g e n of the r a b b i t . The a n t i s e r a r a i s e d a g a i n s t t h i s a n t i g e n r e v e a l e d p a r t i a l i n h i b i t i o n on p o s t - f e r t i l i z a t i o n events but not on i n i t i a l f e r t i l i z a t i o n s t e p s (Naz e t a l , 1984b). Likew i s e , an acrosomal a n t i g e n (RSA-1) of r a b b i t sperm, which was i n v o l v e d i n sperm-egg i n t e r a c t i o n and f e r t i l i z a t i o n was p u r i f i e d (O'Rand e t a l , 1984). A membrane an t i g e n of human and murine sperm, FA-1, i s r e c o g n i z e d by a monoclonal antibody, MA-24 which i n h i b i t s the 16 i n i t i a l f e r t i l i z a t i o n process (Naz e t a l , 1984a and 1986). A sperm s u r f a c e a n t i g e n , PH-20, was p u r i f i e d from the o c t y l g l u c o s e e x t r a c t e d from guinea p i g sperm (Primakoff, 1988a). A c t i v e immunization of t h i s sperm a n t i g e n r e s u l t e d i n a complete and r e v e r s i b l e s u p p r e s s i o n of f e r t i l i t y i n guinea p i g s (Primakoff, 1988b). From numerous p r e v i o u s s t u d i e s by other i n v e s t i g a t o r s , i t i s apparent t h a t the most s u i t a b l e sperm antigens f o r c o n t r a c e p t i v e v a c c i n e s must be s p e r m - s p e c i f i c and r e a d i l y a c c e s s i b l e t o b i n d i n g by IgG or IgA a n t i b o d i e s (Tung, 1980; Anderson and Alexander, 1933). The most d e s i r a b l e ones are those of which the f u n c t i o n can be blocked by bound a n t i b o d i e s t o i n t e r f e r e w i t h the f e r t i l i z a t i o n process.- However, i n f o r m a t i o n concerning the s t r u c t u r a l aspects of sperm s u r f a c e antigens appear t o be very l i m i t e d . T h e r e f o r e , my work not o n l y d e a l t w i t h the b a s i c study of the sperm a n t i g e n , SA-63, but a l s o i t s a p p l i c a t i o n t o the development of an imraunocontraceptive v a c c i n e . 17 I I . MATERIALS AND METHODS (The formula of s o l u t i o n s i n t h i s s e c t i o n are summarized i n Appendix.) IMMUNOLOGICAL TECHNIQUES 1. CHEMICALS F l u o r e s c e i n - i s o t h i o c y a n a t e (FITC), peroxidase (type VI, from H o r s e r a d i s h ) , D i e t h y l a m i n o e t h y l ( D E A E ) - c e l l u l o s e and ammonium s u l f a t e , complete and incomplete Freund's adjuvant were from Sigma ( S t . L o u i s , MO). F I T C - l a b e l e d goat anti-mouse/rabbit IgG+M+A was from GIBCO/BRL ( B u r l i n g t o n , O n t a r i o , Canada). M i c r o t i t e r p l a t e s f o r immunoassays (Immunlon I) were from Dynatech ( A r l i n g t o n , VA). 2. ANIMALS Randomly bred CD-I female mice of 8-10 weeks o l d were used f o r a c t i v e immunization. New Zealand white female r a b b i t s between 2 and 3 months of age were used f o r immunizations. 3. PURIFICATION OF ANTIBODY FROM ASCITES FLUID AND ANTISERA The HS-6 3 monoclonal antibody (IgG 1 s u b c l a s s ) i n t h i s study 18 was s e c r e t e d by a h y b r i d c e l l l i n e generated by means of c e l l f u s i o n s between NS-1 myeloma c e l l s and s p l e e n c e l l s of BALB/C mice immunized wi t h human sperm a n t i g e n p r e p a r a t i o n (Lee e t a l . , 1984a). G e n e r a l l y , HS-63 monoclonal antibody c o u l d be o b tained and p u r i f i e d from the a s c i t e s f l u i d of BALB/C mice which were i n j e c t e d p e r i t o n e a l l y w i t h the e s t a b l i s h e d h y b r i d c e l l s s e c r e t i n g t h i s antibody. The t i t r e s of t h i s antibody i n a s c i t e s f l u i d were determined by i n d i r e c t immunofluorescent assay and u s u a l l y i n the order of 1:100,000 d i l u t i o n . Monoclonal a n t i b o d i e s from a s c i t e s f l u i d or a n t i s e r a were p u r i f i e d by ammonium s u l f a t e f r a c t i o n a t i o n and D E A E - c e l l u l o s e chromatography ( H e i d e and SchwicK, 1973). S u f f i c i e n t ammonium s u l f a t e was added t o the antibody s o l u t i o n ( e i t h e r a s c i t e s f l u i d , c u l t u r e supernatant or a n t i s e r a ) to g i v e a f i n a l c o n c e n t r a t i o n of 313 gram per l i t e r (50 % s a t u r a t i o n ) . The s o l u t i o n was s l o w l y s t i r r e d f o r 30 minutes and c e n t r i f u g e d a t 10,000 X g f o r 10 minutes. The p e l l e t was then recovered and washed at 0°C w i t h a s o l u t i o n of 1.75 M ammonium s u l f a t e u n t i l the s o l u t i o n turned m i l k white (Harboe and I n g i l d , 1973). A f t e r c e n t r i f u g a t i o n , the p e l l e t was d i s s o l v e d i n 10 mM T r i s b u f f e r pH 8.0 and d i a l y z e d o v e r n i g h t a g a i n s t the same b u f f e r . The d i a l y z e d immunoglobulin s o l u t i o n was then loaded to a DEAE column (e.g. 5 ml DEAE-19 c e l l u l o s e per ml of a s c i t e s f l u i d ) which was e q u i l i b r a t e d i n the same b u f f e r . The column was washed with two bed volumes of the b u f f e r and the immunoglobulin was e l u t e d by u s i n g a l i n e a r g r a d i e n t of 0 t o 0.3 M NaCl i n the same b u f f e r . The t o t a l volume of the s a l t g r a d i e n t was 5 t o 7. times of the column bed volume. The immunoglobulin (IgG) was e l u t e d as a sharp peak, and the p u r i t y was t e s t e d by u s i n g SDS-PAGE. With t h i s p u r i f i c a t i o n method, the p u r i f i e d immunoglobulin was u s u a l l y 90 % pure. 4 . LABELING OF ANTIBODY a. IODINATION OF ANTIBODY (Greenwood and Hunter, 1963) Ten ug of p u r i f i e d antibody i n 80 u l of 0.1 M phosphate b u f f e r , pH 7.1 was mixed w i t h 0.5 mCi of " - ' i i n a. t e s t tube. Twenty u l of Chloramine T (2.5 mg/ml) was.then added t o the mixture. A f t e r 30 seconds of i n c u b a t i o n , 100 u l of NaHS0 3 (1.2 mg/ml) was added and the mixture was allowed t o stand f o r an a d d i t i o n a l 3 0 seconds. F i n a l l y , 100 u l of Nal was added t o the r e a c t i o n mixture. Free 1 2 5 i was separated by g e l f i l t r a t i o n on a Sephadex G-25 column (10 ml) which was e q u i l i b r a t e d by the same r e a c t i o n b u f f e r c o n t a i n i n g 0.5 % BSA. The s p e c i f i c r a d i o a c t i v i t y 1 P 5 of the I - l a b e l e d antibody was determined by u s i n g an LKB 20 minigamma counter. b. PREPARATION OF FITC-LABELED ANTIBODY One mg of the p u r i f i e d antibody i n 0.5 ml of 0.1 M NaHC0 3, pH 9.5 was mixed wi t h 12.5 ug F l u o r e s c e i n - i s o t h i o c y a n a t e (FITC). A f t e r o v e r n i g h t shaking a t 4°C i n the dark, the f r e e FITC was separated from the F I T C - l a b e l e d antibody by a Sephadex G-25 g e l f i l t r a t i o n column (10 ml) which was e q u i l i b r a t e d by PBS c o n t a i n i n g 0.5 % BSA. The f i r s t peak which e l u t e d from the Sephadex G-25 column was the F I T C - l a b e l e d antibody. c. PREPARATION OF HORSE RADISH PEROXIDASE (HRP)-LABELED ANTIBODY (Nakane and Kawaoi, 1974) Four mg horse r a d i s h peroxidase i n 1 ml d e i o n i z e d water was incubated with 0.2 ml of 0.2 M NaI0 4 a t room temperature f o r 20 minutes. The r e a c t i o n mixture was then d i a l y z e d a g a i n s t 1 mM sodium a c e t a t e , pH 4.5 a t 4°C f o r 3 hours with 5 changes of b u f f e r every 30 minutes. The d i a l y z e d HRP was incubated with 10 mg of monoclonal antibody i n 2 ml of 0.2 M NaHC0 3, pH 9.5. A f t e r one hour i n c u b a t i o n a t room temperature, the r e a c t i o n was stopped by adding NaBH 4 to a f i n a l c o n c e n t r a t i o n o f 0.2 mg/ml. The HRP-l a b e l e d antibody was d i a l y z e d a g a i n s t PBS ov e r n i g h t a t 4°C and 21 was then s t o r e d i n PBS with 0.5 % BSA and 0.1 % Thimerosal a t 4°C. 5. INDIRECT IMMUNOFLUORESCENT ASSAY The i n d i r e c t immunofluorescent assay was used t o determine the b i n d i n g of a n t i b o d i e s t o sperm s u r f a c e antigens and used e s p e c i a l l y f o r determinations of antibody t i t r e s (Lee e t a l , 1984a,b,c). a. PREPARATION OF SPERM SLIDES Human sperm were ob t a i n e d from the e j a c u l a t e s of h e a l t h y donors. A f t e r 30 minutes f o r l i q u e f a c t i o n , the sperm were washed twice by c e n t r i f u g a t i o n a t 500 X g f o r 10 minutes wi t h 10 volumes of PBS. The washed sperm was a d j u s t e d t o 5 x 10 6/ ml i n PBS and then mounted on s l i d e s . A i r - d r i e d sperm s l i d e s were s t o r e d a t -20°C. Mouse sperm were o b t a i n e d from the cauda e p i d i d y m i d i s and from the vas deferens of mature male mice. The d i s s e c t e d cauda e p i d i d y m i d i s and vas deferens were c u t i n t o s m a l l p i e c e s , and then incubated a t 37°C i n a C 0 2 i n c u b a t o r f o r 15 minutes i n BWW medium. The t i s s u e d e b r i s was removed by a c o t t o n f i b e r column. The sperm which swam out were prepared l i k e human sperm f o r sperm 22 s l i d e s . The acrosome-reacted sperm were prepared f o l l o w i n g the method d e s c r i b e d by J a m i l and White (1981). b. IMMUNOFLUORESCENT ASSAY The s l i d e s o f sperm were f i x e d i n a b s o l u t e methanol f o r 10 minutes a t room temperature, d r i e d , and 0.5 % BSA i n PBS was a p p l i e d f o r 10 minutes t o b l o c k n o n s p e c i f i c b i n d i n g . The sperm s l i d e s were incubated w i t h an antibody s o l u t i o n of s e r i a l d i l u t i o n s i n PBS wit h 0.5 % BSA f o r 30 minutes a t room temperature. A f t e r i n c u b a t i o n , the sperm s l i d e s were washed wi t h PBS twi c e . A f f i n i t y - p u r i f i e d goat anti-mouse o r r a b b i t IgG+M+A conjugated with FITC d i l u t e d i n PBS c o n t a i n i n g 0.5 % BSA was added as the second antibody and incubated f o r an a d d i t i o n a l 30 minutes. F o l l o w i n g the t h r e e washes with PBS s o l u t i o n , the immunofluorescent s t a i n i n g was examined by f l u o r e s c e n c e microscopy. I n d i r e c t immunofluorescent i n h i b i t i o n assay was u s u a l l y used f o r q u a n t i t a t i v e d e t e r m i n a t i o n of immunoactivity of the sperm a n t i g e n . The antibody was f i r s t d i l u t e d t o i t s end p o i n t . The end p o i n t was d e f i n e d as the maximum d i l u t i o n of antibody which s t i l l s t a i n e d the methanol-fixed sperm i n the i n d i r e c t 23 immunofluorescent assay. The end p o i n t of HS-63 from a s c i t e s f l u i d was about 1:100,000. In the i n h i b i t i o n assay, an equal volume of a n t i g e n p r e p a r a t i o n and HS-63 (1:50,000 from a s c i t e s f l u i d ) were incubated a t 37°C f o r one hour. The s o l u t i o n was then a p p l i e d t o the a i r - d r i e d and methanol-fixed sperm f o r h a l f an hour a t room temperature. F o l l o w i n g t h r e e washes with 0.5% bovine serum albumin i n phosphate-buffered s a l i n e (PBS-BSA), FITC-l a b e l e d goat anti-mouse IgG+M was added to sperm as the second antibody. The absence of the immunofluorescent s t a i n i n g of sperm i n d i c a t e d the presence of a n t i g e n . Mouse t e s t e s and l i v e r homogenates served as the p o s i t i v e and negative c o n t r o l s , r e s p e c t i v e l y . An i n d i r e c t immunofluorescent i n h i b i t i o n assay was a l s o used t o determine the t i s s u e - s p e c i f i c i t y of monoclonal a n t i b o d i e s and isoimmune a n t i s e r a a c c o r d i n g to the method of Gaunt (Gaunt, 1982). B r i e f l y , a n t i b o d i e s from e i t h e r a s c i t e s f l u i d o r isoimmune a n t i s e r a ( d i l u t i o n f a c t o r , 1:100 t o .1:10,000) were incubated w i t h an equal volume of f r e s h whole homogenates of t e s t i s , b r a i n , h e a r t , kidney, s p l e e n or l i v e r ( p r o t e i n c o n c e n t r a t i o n of a l l homogenates was a d j u s t e d t o 30 mg/ml). A f t e r one hour i n c u b a t i o n at room temperature, the mixture was c e n t r i f u g e d a t 27,000 x g f o r 10 minutes. The supernatant was recovered f o r the i n d i r e c t 24 immunofluorescent assay with methanol-fixed mouse sperm. 6. DIRECT IMMUNOFLUORESCENT ASSAY The d i r e c t immunofluorescent assay was used t o eva l u a t e the immunofluorescent r e a c t i o n of F I T C - l a b e l e d antibody on l i v e sperm. P r e p a r a t i o n of l i v e sperm i s d e s c r i b e d i n s e c t i o n D.3.b. from mice and i n s e c t i o n D.4.b. from humans. 50 u l of l i v e sperm were incubated with 50 u l of FITC l a b e l e d HS-63 which was d i l u t e d i n PBS i n a 1.5 ml m i c r o c e n t r i f u g e tube f o r 1 hour a t 37°C. A f t e r i n c u b a t i o n , the immunofluorescent s t a i n i n g was examined by f l u o r e s c e n c e microscopy. In the experiment of c h e m i c a l l y t r e a t e d l i v e sperm, 50 u l of l i v e sperm were incubated s e p a r a t e l y w i t h equal volumes of 1 M KC1, 0.4 % g l u t a r a l d e h y d e or 0.2 % T r i t o n X-100 which were prepared i n BWW medium f o r 5 minutes a t room temperature. A f t e r c e n t r i f u g a t i o n , the sperm were resuspended i n 50 u l of BWW medium and then incubated with FITC l a b e l e d HS-63 as d e s c r i b e d p r e v i o u s l y . 7. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) (The formular of s o l u t i o n s are summarized i n Appendix 1) 25 a. PREPARATION OF ANTIGEN-COATED MICROWELLS P u r i f i e d a n t i g e n (about 1 ug/ml), the s o l u b l e e x t r a c t of human or mouse sperm i n 0.1 M T r i s - H C l b u f f e r , pH 8.0 was coated on m i c r o t i t e r p l a t e s o v e r n i g h t a t 4°C. A f t e r t h r e e washes wi t h d i s t i l l e d water and b l o c k i n g w i t h b l o c k i n g b u f f e r f o r 1 hour, the coated m i c r o t i t e r p l a t e s were d r i e d a t room temperature and were i s t o r e d a t 4°C. Prepared m i c r o w e l l s can be kept a t 4°C f o r s e v e r a l months. b. ASSAY PROCEDURES One hundred u l of HS-63 monoclonal antibody, isoimmune s e r a or other u n r e l a t e d a n t i b o d i e s i n a s e r i e s of f i v e or t e n f o l d d i l u t i o n s were incubated with a n t i g e n - c o a t e d m i c r o w e l l s f o r a g i v e n p e r i o d of time a t room temperature. The unbound a n t i b o d i e s were removed by washing t h r e e times with 0.02 % Tween-20 i n PBS (PBS-Tween). Goat anti-mouse IgG+M l a b e l e d w i t h horse r a d i s h peroxidase (from BRL) a t a d i l u t i o n o f 1:3,000 i n PBS with 0.5 % BSA was added f o r another 1 hour of i n c u b a t i o n a t 37°C. A f t e r t h r e e washes with PBS-Tween and one wash with water, the antibody b i n d i n g was v i s u a l i z e d by the c o l o r i m e t r i c enzymatic r e a c t i o n i n the presence of 200 u l of s u b s t r a t e s o l u t i o n . A f t e r c o l o r development i n the dark f o r a s u i t a b l e p e r i o d of time, the 26 r e a c t i o n was stopped by adding an equal volume of 1 M H 2S0 4- The r e s u l t was measured by a m i c r o t i t e r p l a t e reader a t 450 nm. 8. RADIOIMMUNOSORBENT ASSAY (RISA) The s p e c i f i c i t y of p u r i f i e d sperm or t e s t i s a n t i g e n s and t h e i r c r o s s - r e a c t i v i t y w i t h other a n t i b o d i e s were e v a l u a t e d by ELISA and RISA (Lee e t a l , 1984a; E n g v a l l and Perlmann, 1971). For the RISA, p u r i f i e d HS-63 monoclonal antibody was l a b e l e d w i t h 1 2 5 I a c c o r d i n g t o the p r e v i o u s l y d e s c r i b e d procedure of p r o t e i n i o d i n a t i o n . The dose-dependent antigen-antibody b i n d i n g assays were performed by i n c u b a t i n g l a b e l e d HS-6 3 of s e r i a l d i l u t i o n s w i t h antigen-coated m i c r o w e l l s a t room temperature f o r one hour. F o l l o w i n g t h r e e washes wi t h PBS-Tween, the r e s i d u a l r a d i o a c t i v i t y i n m i c r o w e l l s was determined by an LKB minigamma counter. The RISA was a l s o used to demonstrate s p e c i f i c i n h i b i t i o n of HS-63 b i n d i n g to sperm coated on m i c r o w e l l s by the a n t i g e n s o l u t i o n . 9. PRODUCTION OF POLYCLONAL ANTISERA The p u r i f i e d sperm a n t i g e n (MSA-63) from mouse was used t o isoimmunize mice and r a b b i t s . B r i e f l y , 10 ug of p u r i f i e d a n t i g e n 27 i n 50 u l PBS were e m u l s i f i e d w i t h an equal volume of complete Freund's adjuvant and i n j e c t e d subcutaneously i n t o each mouse f o r the primary immunization. Subsequent immunizations (10 ug/mouse) were performed a t biweekly i n t e r v a l s u s i n g incomplete Freund's adjuvant. Seven days a f t e r each immunization, animals were b l e d and the a n t i s e r a t i t r e s were determined by the i n d i r e c t immunofluorescent assay. Rabbits were immunized with the same p r o t o c o l , except t h a t 50 ug of a n t i g e n was used f o r each immunization. The t i t r e and s p e c i f i c i t y o f the a n t i s e r a from mouse or r a b b i t were determined by e i t h e r immunofluorescent assay or by ELISA. 28 C. PROTEIN CHEMISTRY 1. CHEMICALS Sephacryl S-300 and Ampholine were from Pharmacia/LKB (Piscataway, NJ). T r y p s i n was from Sigma (St. L o u i s , MO). A f f i -g e l 10, p r o t e i n assay reagent and a l l the a n a l y t i c a l grade reagents r e q u i r e d f o r 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 were from Bio-Rad (Richmond, CA). N i t r o c e l l u l o s e f i l t e r paper was from M i l l i p o r e ( M i s s i s s a u g a , O n t a r i o , Canada). l 2 5 i ( i mCi/mmol) was from Amersham ( O a k v i l l e , O n t a r i o , Canada). N - g l y c o s i d a s e and O-g l y c o s i d a s e were from Boehringer Mannheim Biochemica ( D o r v a l , Quebec, Canada). C e n t r i p r e p 10 and C e n t r i c o n 10 c o n c e n t r a t o r was from Amicon (Danvers, MA). 2. DETERMINATION OF PROTEIN CONCENTRATION The p r o t e i n c o n c e n t r a t i o n was r o u t i n e l y estimated by measuring o p t i c a l d e n s i t y (OD) a t 280 nm. However, Bio-Rad dye reagent was used to determine the p r o t e i n c o n c e n t r a t i o n i n m i c r o s c a l e (Bradford, 1976). In t h i s method, 0.2 ml of Bio-Rad s t a i n was added t o 0.8 ml of sample s o l u t i o n c o n t a i n i n g 1 t o 10 ug p r o t e i n . The p r o t e i n c o n c e n t r a t i o n was determined by r e a d i n g the OD a t 595 nm a f t e r 2 minutes. Bovine serum albumin was used 29 as a standard. Amido b l a c k s t a i n i n g method was used t o determine the p r o t e i n c o n c e n t r a t i o n i n sample s o l u t i o n s c o n t a i n i n g detergent ( S c h a f f n e r and Weissman, 1973). 3. DETECTION OF ANTIGEN IMMUNOACTIVITY The i n d i r e c t immunofluorescent i n h i b i t i o n assay and RISA were used t o determine a n t i g e n immunoactivity d u r i n g v a r i o u s p u r i f i c a t i o n s t a g e s. The r e l a t i v e s p e c i f i c immunoactivity of p u r i f i e d a n t i g e n was d e f i n e d as the i n v e r s e of p r o t e i n c o n c e n t r a t i o n of a g i v e n a n t i g e n p r e p a r a t i o n t h a t was r e q u i r e d t o i n h i b i t the immunofluorescent s t a i n i n g of sperm by HS-63 at i t s end p o i n t d i l u t i o n . For the i n d i r e c t immunofluorescent i n h i b i t i o n assay, HS-6 3 a s c i t e s f l u i d which had been d i l u t e d t o 1:10,000 wi t h PBS-BSA, was incubated with an equal volume of a n t i g e n s o l u t i o n f o r 1 hour a t 37°C. The procedures of i n d i r e c t immunofluorescent assay was d e s c r i b e d p r e v i o u s l y i n the s e c t i o n of immunofluorescent assay. 4. PREPARATION OF IMMUNOAFFINITY GEL P u r i f i e d HS-63 monoclonal antibody (50 mg i n 50 ml of 0.1 M NaHCOo) was mixed with 5 ml A f f i - g e l 10 which had been washed 30 w i t h d i s t i l l e d water f o r the removal of i s o p r o p a n o l i n the g e l suspension. A f t e r o v e r n i g h t shaking a t 4°C, the g e l was washed with water and then incubated w i t h 1 M T r i s - H C l , pH 8.0 f o r 1 hour a t room temperature. F i n a l l y , the g e l was washed wi t h a s o l u t i o n o f 6 M urea and 2 M NaCl and e q u i l i b r a t e d w i t h PBS. The coupled g e l was s t o r e d w i t h 0.01 % NaN 3 a t 4°C . 5. PURIFICATION OF MSA-63 ANTIGEN FROM MOUSE TESTES Sperm or t e s t i s a n t i g e n s p e c i f i c a l l y r e c o g n i z e d by HS-63 was p u r i f i e d from mouse t e s t e s by u s i n g two d i f f e r e n t procedures d e s c r i b e d as f o l l o w s : a. CONVENTIONAL METHOD COMBINED WITH IMMUNOAFFINITY CHROMATOGRAPHY Ten grams of f r o z e n t e s t e s were homogenized i n 100 ml PBS c o n t a i n i n g 1 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF) wi t h a p o l y t r o n homogenizer (Brinkmann) a t 4°C. A f t e r c e n t r i f u g a t i o n a t 27,000 X g f o r 20 minutes, the supernatant was c o l l e c t e d and s u b j e c t e d t o ammonium s u l f a t e f r a c t i o n a t i o n . In t h i s procedure, s o l i d ammonium s u l f a t e (390 mg/ml) was s l o w l y added t o the supernatant, and then s t i r r e d f o r 1 hour a t 4°C. A f t e r c e n t r i f u g a t i o n again, the p e l l e t was d i s s o l v e d i n 50 mM phosphate 31 b u f f e r (potassium s a l t ) pH 7.2 and d i a l y z e d o v e r n i g h t a g a i n s t the same b u f f e r . The d i a l y z a t e was then a p p l i e d onto a D E A E - c e l l u l o s e column (2.5 x 40 cm) which had been e q u i l i b r a t e d w i t h the same phosphate b u f f e r . The adsorbed p r o t e i n s were e l u t e d w i t h a 0 t o 1 M NaCl l i n e a r g r a d i e n t (500 ml:500 ml). The a n t i g e n c o n t a i n i n g f r a c t i o n s d e t e c t e d by u s i n g i n d i r e c t immunofluorescent i n h i b i t i o n assay were pooled and d i a l y z e d a g a i n s t PBS o v e r n i g h t . The s o l u t i o n was then loaded on an immunoaffinity column (10 ml) us i n g p u r i f i e d HS-63 monoclonal antibody as the a f f i n i t y l i g a n d . A f t e r the a p p l i c a t i o n of the sample, the column was washed e x t e n s i v e l y with 0.5 M NaCl i n PBS u n t i l adsorbance of the e l u e n t at 280 nm was 0.001 or l e s s . The a n t i g e n was e l u t e d w i t h 0.1 M g l y c i n e - H C l b u f f e r a t pH 2.2. The f r a c t i o n s c o n t a i n i n g most of the a n t i g e n immunoactivity were pooled, and con c e n t r a t e d by c e n t r i f u g a t i o n u s i n g Centricon-10. b. IMMUNOAFFINITY CHROMATOGRAPHY WITH DETERGENT EXTRACTION Ten grams of mouse t e s t e s were homogenized i n PBS with 2 % T r i t o n X-100 and 2 mM PMSF a t 4°C. A f t e r c e n t r i f u g a t i o n a t a speed of 27,000 X g f o r 10 minutes, the supernatant was c o l l e c t e d and passed through a column of c o t t o n f i b e r s t o remove the t i s s u e d e b r i s . The supernatant was d i l u t e d t o 100 ml and then a p p l i e d t o 32 an immunoaffinity g e l (10 ml) u s i n g HS-63 as the a f f i n i t y l i g a n d . A f t e r repeated l o a d i n g s f o r 2 hours a t 4°C, the a f f i n i t y g e l was washed wit h 500 ml of PBS c o n t a i n i n g 0.5 M NaCl and 0.1% T r i t o n X-100. The bound a n t i g e n was then e l u t e d w i t h 0.1 M g l y c i n e - H C l , pH 2.2 c o n t a i n i n g 0.1 % T r i t o n X-100 and 2 mM PMSF. The a n t i g e n -c o n t a i n i n g f r a c t i o n s were pooled, and conc e n t r a t e d by c e n t r i f u g a t i o n u s i n g a C e n t r i p r e p c o n c e n t r a t o r . 6 . GEL FILTRATION Sephacryl S-300 g e l f i l t r a t i o n chromatography was used t o determine the n a t i v e molecular weight of sperm a n t i g e n i n the p u r i f i e d s t a t e and i n the homogenate. Swollen Sephacryl S-300 was packed i n t o a 30 cm X 0.9 cm g l a s s column and was e q u i l i b r a t e d with PBS c o n t a i n i n g 0.1 % T r i t o n X-100. Supernatant of t e s t e s homogenate or p u r i f i e d MSA-63 an t i g e n i n a volume of 0.5 ml c o n t a i n i n g 10 % g l y c e r o l was loaded on to the column. At the same time, f r a c t i o n s of 1 ml were c o l l e c t e d . The immunoactivity of a n t i g e n was d e t e c t e d by u s i n g the i n d i r e c t i n h i b i t i o n immunofluorescent assay as d e s c r i b e d above. 33 7. SODIUM DEDECYL SULFATE (SDS) POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) (The formular of s o l u t i o n s are summarized i n Appendix 2) A. PROCEDURE OF ELECTROPHORESIS A v e r t i c a l s l a b g e l apparatus f o r SDS-PAGE was o b t a i n e d from Albergene Co. G e n e r a l l y , 3 % acrylamide s t a c k i n g g e l and 10 % acrylamide running g e l were used (Laemmli, 1970). The running g e l was prepared from a mixture c o n t a i n i n g 10 ml running g e l acrylamide s o l u t i o n , 7.5 ml running T r i s b u f f e r , 12 ml water, 0.5 ml of 2 % potassium p e r s u l f a t e and 25 u l N,N,N',N'-tetramethylenediamine (TEMED). Immediately a f t e r mixing, the running g e l was f i l l e d between the g l a s s p l a t e s . t o a h e i g h t of 10 cm, water was o v e r l a i d on top of the g e l t o ensure a l e v e l s u r f a c e f o r the running g e l . S t a c k i n g g e l was prepared from a mixture c o n t a i n i n g 1 ml s t a c k i n g g e l acrylamide s o l u t i o n , 2.5 ml s t a c k i n g T r i s b u f f e r , 6 . 3 4 ml water, 0.1 ml of 2 % potassium p e r s u l f a t e and 5 u l TEMED. When the running g e l was formed, the water on top of the g e l was d i s c a r d e d . The s t a c k i n g g e l was o v e r l a i d on top of the running g e l . A s l o t comb was p l a c e d a t the upper margin of g l a s s p l a t e t o form w e l l s f o r the l o a d i n g of the p r o t e i n samples. The p r o t e i n sample was mixed with equal volumes of sample 34 b u f f e r , b o i l e d f o r 5 minutes t o denature' the p r o t e i n s , and then a p p l i e d t o the g e l . The v o l t a g e of e l e c t r o p h o r e s i s was f i r s t s e t a t 150 V. A f t e r the p r o t e i n had entered the running g e l , the v o l t a g e was then switched t o 250 V. The e l e c t r o p h o r e s i s was run u n t i l the dye f r o n t reached about 10 cm from the top of running g e l . A f t e r the e l e c t r o p h o r e s i s , the g e l was removed from the g l a s s p l a t e s c a r e f u l l y f o r p r o t e i n s t a i n i n g or f o r Western b l o t a n a l y s i s . b. PROTEIN STAINING BY COOMASSIE BRILLIANT BLUE For Coomassie b r i l l i a n t b l u e s t a i n i n g , the g e l was immersed i n a s o l u t i o n c o n t a i n i n g 0.2 % Coomassie b r i l l i a n t blue R i n 10 % a c e t i c a c i d , 50 % methanol, and 40 % dH 20 f o r 4 hours or more. The g e l was d e s t a i n e d by repeated washings i n 50 % methanol, 10 % a c e t i c a c i d , and 40 % dH 20 u n t i l the p r o t e i n bands on the g e l were v i s u a l i z e d . F i n a l l y , the g e l was kept i n 10 % a c e t i c a c i d . c. PROTEIN STAINING BY SILVER REAGENT S i l v e r s t a i n i n g k i t from Bio-RAD was used f o r p r o t e i n s t a i n i n g . A f t e r e l e c t r o p h o r e s i s , the g e l was f i x e d i n a s o l u t i o n o f 40 % methanol and 10 % a c e t i c a c i d f o r 30 minutes f o l l o w e d by 35 a s o l u t i o n c o n t a i n i n g 10 % ethanol and 5 % a c e t i c a c i d f o r two changes a t 30 minutes i n t e r v a l . A f t e r i n c u b a t i o n with o x i d i z e r f o r 5 minutes, the g e l was washed w i t h dH 20 f o r t h r e e changes of 5 minutes. The g e l was then incubated w i t h s i l v e r reagent f o r 20 minutes. A f t e r a r i n s e i n dH 20, developer s o l u t i o n was added and the g e l was incubated f o r 30 seconds or u n t i l the s o l u t i o n t u r n e d dark. The p r o t e i n bands on the g e l were v i s u a l i z e d by c o n t i n u a l i n c u b a t i o n i n the developer s o l u t i o n . The s t a i n e d g e l can be s t o r e d i n 5 % a c e t i c a c i d s o l u t i o n f o r up t o s e v e r a l months. 8 . TWO-DIMENSIONAL GEL ELECTROPHORESIS (The formular of s o l u t i o n s are summarized i n Appendix 3) Two-dimensional g e l e l e c t r o p h o r e s i s was performed by f i r s t r unning i s o e l e c t r i c f o c u s i n g g e l e l e c t r o p h o r e s i s and then by s l a b SDS-PAGE a c c o r d i n g to the p u b l i s h e d procedures ( O ' F a r r e l l e t a l . , 1977). For the i s o e l e c t r i c f o c u s i n g g e l e l e c t r o p h o r e s i s , f i v e ml of g e l mixture was made and f i l l e d i n t o a 10 cm g l a s s tube (made from a 1 ml s e r o l o g i c a l p i p e t t e ) . A l a y e r of water was c a r e f u l l y o v e r l a i d on top of the g e l . 50 u l of p u r i f i e d p r o t e i n was mixed with sample b u f f e r of the same volume. 0.2 % H 3 P 0 4 was the anode b u f f e r and 0.1 % NaOH was the cathode b u f f e r . The e l e c t r o p h o r e s i s was c a r r i e d out a t a v o l t a g e of 350 V f o r o v e r n i g h t and then 500 V f o r 1 hour a t room temperature. A f t e r the e l e c t r o p h o r e s i s , the g e l was removed and immersed i n a SDS sample b u f f e r f o r 1 hour i n order t o remove urea from the g e l . The g e l was then p l a c e d on t o p of a s l a b SDS g e l . The two g e l s were s e a l e d t o g e t h e r b y u s i n g 1 % agarose. The second dimension SDS-PAGE was performed on g e l s l a b s as p r e v i o u s l y d e s c r i b e d . 9. IMMUNOBLOTTING ANALYSIS a. DOT BLOT ASSAY (The formular of s o l u t i o n s are summarized i n Appendix 4) The dot b l o t assay was m o d i f i e d from the method d e s c r i b e d by Towbin (Towbin e t a l . , 1979). T h i s method g i v e s r a p i d d e t e c t i o n of a n t i g e n i n v a r i o u s samples and i s e s p e c i a l l y u s e f u l f o r t e s t i n g column e l u e n t s d u r i n g a n t i g e n p u r i f i c a t i o n . F i v e u l of sample was d o t t e d onto n i t r o c e l l u l o s e f i l t e r paper and allowed t o dry completely. The n i t r o c e l l u l o s e f i l t e r was t r e a t e d w i t h b l o c k i n g s o l u t i o n f o r 30 minutes and then incubated w i t h r a b b i t anti-MSA-63 s e r a w i t h a d i l u t i o n of 1 t o 200 f o r 1 hour at room temperature. A f t e r washing t h r e e times w i t h TBST HRP-labeled goat a n t i - r a b b i t IgG+M antibody (from BRL) with a d i l u t i o n of 1 t o 200 was added and incubated f o r 1 hour. F o l l o w i n g t h r e e washes with TBST and once wi t h TBS, the n i t r o c e l l u l o s e f i l t e r paper was incubated w i t h a peroxidase s u b s t r a t e s o l u t i o n f o r c o l o r development u n t i l c l e a r s i g n a l appeared. The c o l o r development was then stopped by water. Dot b l o t assay was a l s o used f o r s t a b i l i t y e v a l u a t i o n of the HS-6 3 e p i t o p e . The supernatant of mouse t e s t e s homogenate was t r e a t e d by the f o l l o w i n g enzymes and chemicals: (a) 2 % SDS, (b) 2 % T r i t o n X-100, ( c ) 0 . 4 % NP-40, (d) 0.2 % t r y p s i n , (e) 0.2 % pronase, ( f ) 40 mM sodium p e r i o d a t e , and (g) 0.2 u n i t / m l neuraminidase. A f t e r i n c u b a t i o n at 37°C f o r one hour, the t r e a t e d supernatant was d o t t e d on a n i t r o c e l l u l o s e f i l t e r and the s t a b i l i t y of the HS-63 epitope was analyzed by u s i n g HRP-labeled HS-6 3 monoclonal antibody (0.5 mg/ml) as a d e t e c t i n g probe. b. WESTERN BLOT ASSAY (The formular of s o l u t i o n s are summarized i n Appendix 5) T h i s method was used t o i d e n t i f y the s p e c i f i c p r o t e i n bands separated by SDS-PAGE i n the presence of a n t i b o d i e s . The d e t a i l s of t h i s method are d e s c r i b e d i n the i n s t r u c t i o n manual s u p p l i e d by BIO-RAD (Towbin e t a l . , 1979). A f t e r e l e c t r o p h o r e s i s , the g e l and n i t r o c e l l u l o s e f i l t e r 38 paper were r i n s e d i n t r a n s f e r b u f f e r . The n i t r o c e l l u l o s e f i l t e r paper was p l a c e d a g a i n s t the g e l on a f l a t s u r f a c e and was l a i d smoothly over the g e l by r o l l i n g a 5-ml g l a s s p i p e t t e on top to remove a l l a i r bubbles. Two p i e c e s of 3 MM f i l t e r paper and sponge (prewetted with t r a n s f e r b u f f e r ) were used t o clamp the g e l / f i l t e r t o make a sandwich. The whole sandwich u n i t was p l a c e d i n e l e c t r o p h o r e t i c t r a n s f e r apparatus w i t h the g e l f a c i n g the cathode. The b u f f e r tank was f i l l e d w i t h t r a n s f e r b u f f e r t o cover the g e l . A f t e r 2 hours of e l e c t r o p h o r e t i c t r a n s f e r a t a constant v o l t a g e of 50 V, the n i t r o c e l l u l o s e f i l t e r paper was p l a c e d i n b l o c k i n g b u f f e r f o r immunoblotting a n a l y s i s which was d e s c r i b e d p r e v i o u s l y . The p r o t e i n p a t t e r n can be v i s u a l i z e d by u s i n g amido bla c k s t a i n i n g . 10. DEGLYCOSYLATION OF PROTEIN D e g l y c o s y l a t i o n of MSA-63 p r o t e i n u s i n g N - g l y c o s i d a s e and O-g l y c o s i d a s e was m o d i f i e d from the i n s t r u c t i o n manual s u p p l i e d with the enzymes and the method d e s c r i b e d by Steube e t a l . (1985). One u l of i o d i n a t e d MSA-63 p r o t e i n ( s p e c i f i c a c t i v i t y 0.1 mCi/ug) was d i l u t e d w i t h 22.5 u l of 0.1 M phosphate b u f f e r pH 8.3 c o n t a i n i n g 1 % T r i t o n X-100 and 0.1 % SDS and then incubated with 39 N - g l y c o s i d a s e (0.4 u n i t ) and 0 - g l y c o s i d a s e (1 mU) , r e s p e c t i v e l y . A f t e r o v e r n i g h t i n c u b a t i o n a t 37°C, the r e a c t i o n mixtures were analyzed by SDS-PAGE. HCG p r o t e i n served as a p o s i t i v e c o n t r o l . 11. AMINO ACID SEQUENCE ANALYSIS A f t e r the e l e c t r o t r a n s f e r , the p r o t e i n spot of the p u r i f i e d MSA-63 a n t i g e n on the 2D-gel was c u t from n i t r o c e l l u l o s e f i l t e r paper which had been s t a i n e d with amido b l a c k . The p r o t e i n spot (about 10 ug) was p l a c e d i n a 1.5 ml c e n t r i f u g e tube and incubated w i t h 0.1 M A c e t i c a c i d c o n t a i n i n g 1 % PVP-40 at 37°C f o r 30 minutes. A f t e r r i n s i n g ten times with water and once with 0.1 M NaHC0 3 pH 8.2, 50 u l of 0.1 M NaHC0 3 c o n t a i n i n g 5% a c e t o n i t r i l e and 2 u l of t r y p s i n (1 mg/ml) were added f o r o v e r n i g h t t r y p t i c d i g e s t i o n at 37°C. F o l l o w i n g d i g e s t i o n , p e p t i d e fragments were separated by h i g h performance l i q u i d chromatography (HPLC) wi t h C-18 r e v e r s e phase column ( M i l l i p o r e Co.) and i n d i v i d u a l l y analyzed u s i n g the method of Edman degradation by a p r o t e i n sequenator ( A p p l i e d Biosystems I n c . ) . 40 D. EVALUATION OF ANTIBODIES BY SPERM FUNCTIONAL ASSAYS The a n t i s e r a were h e a t - i n a c t i v a t e d a t 56 C f o r 45 minutes p r i o r t o use f o r the sperm f u n c t i o n a l assay. The a n t i f e r t i l i t y e f f e c t s of these isoimmune a n t i s e r a on mouse i n v i t r o f e r t i l i z a t i o n (Lee e t a l , 1985; and Biggers e t a l , 1971) and human sperm p e n e t r a t i o n of zona-free hamster eggs (Yanagimachi, 1976) were e v a l u a t e d . HS-63 monoclonal antibody and normal mouse or r a b b i t s e r a served as the p o s i t i v e and the n e g a t i v e c o n t r o l f o r each experiment, r e s p e c t i v e l y . Monoclonal a n t i b o d i e s , MS-204 and MS-207 generated a g a i n s t mouse sperm were served as p o s i t i v e c o n t r o l i n i n v i v o mating study. 1. CHEMICALS Calcium ionophore-A23187, Pisum sativum l e c t i n (PSA), pregnant mare's serum gonadotropin (PMSG), human c h o r i o n i c gonadotropin (hCG), bovine "serum albumin (BSA), lacmoid, h y a l u r o n i d a s e and t r y p s i n were from Sigma (St. L o u i s , MO). Chemicals f o r c u l t u r e media were t i s s u e c u l t u r e grade from GIBCO/BRL ( B u r l i n g t o n , O n t a r i o ) . 2 . ANIMALS Randomly bred CD-I female mice (4 and 6 weeks old) were used 41 f o r i n v i v o mating, i n v i t r o f e r t i l i z a t i o n and a c t i v e immunization. Male mice of CD-I s t r a i n (8 and 10 weeks o l d ) were used f o r mating experiments a f t e r they had been proven to be f e r t i l e . Inbred male BALB/c mice were used f o r a s c i t e s f l u i d p r o d u c t i o n . Eight-week-old female golden hamsters were used f o r the human sperm p e n e t r a t i o n assay. New Zealand white female r a b b i t s of 2 t o 3 months o l d were used f o r immunization. 3. IN VITRO FERTILIZATION OF MOUSE OOCYTES (Lee e t al. , , 1985, •• Biggers e t a l . , 1971). (The formular of s o l u t i o n s are summarized i n Appendix 6) a. INDUCTION OF SUPEROVULATION Female mice were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 5 IU PMSG a t 6 pm on day 1 and f o l l o w e d by a second i n j e c t i o n with 5 IU hCG a t 6 pm on day 3. The l i g h t c y c l e of the animal room was s e t from 5 am t o 7 pm. b. SPERM PREPARATION On day 4, two male mice were k i l l e d a t 8 am. The cauda e p i d i d y m i d i s and vas deferens were d i s s e c t e d out and p l a c e d i n t o a s t e r i l i z e d p e t r i d i s h with 2 ml BWW medium. Sperm were r e l e a s e d 42 by c u t t i n g the t i s s u e s i n t o s m a l l p i e c e s . A f t e r 10 minutes of i n c u b a t i o n a t 37°C i n a C 0 2 i n c u b a t o r , the sperm suspension was passed through a c o t t o n f i b e r column t o remove the t i s s u e d e b r i s . The sperm suspension was then incubated a t 37°C i n a C0 2 i n c u b a t o r f o r 1 hour t o ensure sperm were f u l l y c a p a c i t a t e d i n B i g g e r s , Whitten and Whittingham (BWW) medium. c. SPERM-EGG INTERACTION About 30 minutes a f t e r the sperm suspension was prepared, female mice were k i l l e d and the o v i d u c t s were d i s s e c t e d out. Oocytes surrounded with cumulus c e l l s were r e l e a s e d by punching h o l e s i n the ampulla of the o v i d u c t with a 30 G needle under a d i s s e c t i n g microscope. For each experiment, 20 u l sperm (1 x 10 sperm/ml) suspension and 20 u l antibody s o l u t i o n of known c o n c e n t r a t i o n were added t o 140 u l of BWW medium p l a c e d under mineral o i l i n 35 mm p e t r i d i s h e s . Eggs with cumulus c e l l s were added to the sperm-antibody mixture. A f t e r a 6 t o 8 hour i n c u b a t i o n i n a C0 2 i n c u b a t o r , the eggs were washed t h r e e times by u s i n g PBS and then f i x e d w i t h 2 % g l u t a l d e h y d e s o l u t i o n o v e r n i g h t . The oocytes were s t a i n e d by s t a i n i n g s o l u t i o n and then t r e a t e d with d e s t a i n i n g s o l u t i o n . The f e r t i l i z a t i o n of oocytes was judged by the 43 appearance of two p r o n u c l e i , two p o l a r bodies, a swollen sperm head, or a sperm t a i l . 4. SPERM PENETRATION TO ZONA-FREE HAMSTER OVA (Yanagimachi, 1976) a. INDUCTION OF SUPEROVULATION Ten days be f o r e the assay, the v a g i n a l d i s c h a r g e of each hamster was checked t o determine the exact stage of t h e i r o e strous c y c l e . Hamsters which were on day 1 of the c y c l e with post o v u l a t o r y mucus were i n j e c t e d i n t r a p e r i t o n e a l l y with 40 IU PMSG a t 9 am on day 1 f o l l o w e d by a 40 IU hCG i n j e c t i o n a t 4:30 pm on day 3. b. PREPARATION OF HUMAN SPERM Human semen was donated by masturbation i n the aft e r n o o n of day 3 from a h e a l t h y donor. A f t e r a l l o w i n g 30 t o 60 minutes f o r complete l i q u e f a c t i o n , one ml of semen was p l a c e d i n a 15 ml s t e r i l e c u l t u r e tube and g e n t l y o v e r l a i d with 2 ml BWW medium. The tubes were t i l t e d a t a 20° angle from the h o r i z o n t a l p o s i t i o n t o maximize the s u r f a c e of the semen-medium i n t e r f a c e . They were then incubated a t 37°C f o r a p e r i o d of 1 hour i n a C 0 2 i n c u b a t o r . A f t e r the i n c u b a t i o n , the c u l t u r e tubes were g e n t l y r e t u r n e d 44 t o the v e r t i c a l p o s i t i o n f o r 15 minutes a t room temperature and the t o p 80 % of the o v e r l a y i n g medium was t r a n s f e r r e d i n t o another s t e r i l e c u l t u r e tube. The sperm were c o l l e c t e d by c e n t r i f u g a t i o n a t 500 X g f o r 5 minutes. The p e l l e t was then resuspended i n BWW medium with a f i n a l c o n c e n t r a t i o n of 10 x 10 6 sperm/ml and incubated o v e r n i g h t i n a C 0 2 i n c u b a t o r . The c o n c e n t r a t i o n of sperm was f i n a l l y a d j u s t e d t o 3.5 x 10 6 sperm/ml. A s o l u t i o n mixture c o n t a i n i n g 10 u l of antibody p r e p a r a t i o n and 100 u l of sperm suspension was then p l a c e d under p a r a f f i n o i l i n a 35 mm p e t r i d i s h . c. SPERM-EGG INTERACTION In the morning of day 4, hamsters were k i l l e d and eggs were f l u s h e d out from the o v i d u c t by u s i n g a s y r i n g e w i t h a 30 G needle. The cumulus c e l l s surrounding oocytes were removed by the a d d i t i o n of 0.1 % h y a l u r o n i d a s e and the zona p e l l u c i d a e were d i g e s t e d by u s i n g 0.1 % t r y p s i n . About t h i r t y zona-free hamster eggs were p i p e t t e d i n t o the sperm-antibody mixture under p a r a f f i n o i l i n p e t r i d i s h e s . The mixture was incubated a t 37°C f o r 3 hours i n a C 0 2 i n c u b a t o r . A f t e r i n c u b a t i o n , the eggs were removed and washed t o remove the l o o s e l y a t t a c h e d sperm from the egg s u r f a c e . The percentage of eggs pen e t r a t e d by sperm was recorded 45 under a phase c o n t r a s t microscope by o b s e r v i n g the presence of a swollen sperm head i n s i d e the zona-free eggs. 5. INHIBITION TO ACROSOME REACTION Mouse sperm were prepared as d e s c r i b e d i n the s e c t i o n on mouse i n v i t r o f e r t i l i z a t i o n . The sperm acrosome r e a c t i o n c o u l d be induced by zona p e l l u c i d a (Lakoski e t a l . , 1988). To examine the zona p e l l u c i d a - i n d u c e d AR, mouse ova were r e t r i e v e d from su p e r o v u l a t e d mice as d e s c r i b e d i n the p r e v i o u s s e c t i o n . Cumulus c e l l s were removed by treatment with 0.1 % h y a l u r o n i d a s e i n PBS. The zona p e l l u c i d a of oocytes were s o l u b i l i z e d i n 0.1 M phosphate b u f f e r , pH 2.5. The s o l u t i o n was then n e u t r a l i z e d with 10 X PBS. The c o n c e n t r a t i o n of zona p e l l u c i d a was a d j u s t e d t o 40 zona/ul. Ten u l of mouse sperm a t a c o n c e n t r a t i o n of 1 X 1 0 6 sperm/ml was incubated with 1 u l of s o l u b i l i z e d zona p e l l u c i d a t o induce acrosome r e a c t i o n . A f t e r 30 minutes of i n c u b a t i o n a t 37°C i n a C 0 2 i n c u b a t o r , the sperm were coated on s l i d e s . F I T C - l a b e l e d Pisum sativum l e c t i n was used as a probe t o estimate the percentage of spermatozoa with i n t a c t sperm acrosome (Cross e t a l . , 1986). To t e s t the e f f e c t of s e l e c t e d monoclonal a n t i b o d i e s on the induced AR, monoclonal antibody of known d i l u t i o n was mixed with 46 the sperm s o l u t i o n a t a r a t i o of 1:10 f o r 30 minutes p r i o r t o the treatment of s o l u b i l i z e d zona p e l l u c i d a . I ncubation w i t h u n r e l a t e d a n t i b o d i e s served as the n e g a t i v e c o n t r o l i n the experiment. 6. IN VIVO MATING EXPERIMENTS Female mice were supe r o v u l a t e d w i t h 5 IU of PMSG on day 1 between 1 and 2 pm. H e a t - i n a c t i v a t e d a s c i t e s f l u i d c o n t a i n i n g a known amount of g i v e n monoclonal sperm antibody was i n j e c t e d i n t r a p e r i t o n e a l l y between 9 and 10 am on days 2 and 3. F i v e IU of hCG was a d m i n i s t e r e d between 1 and_ 2 pm on day 3 f o l l o w e d by mating with proven f e r t i l e males. In the morning of day 4, the v a g i n a l plugs of females were checked f o r evidence of mating. In the morning of day 5, a n t i b o d y - t r e a t e d female mice were s a c r i f i c e d , and percentages of r e c o v e r e d embryos i n the t w o - c e l l stage were recorded. •7. STATISTICAL ANALYSIS Student's t t e s t or Chi-square t e s t was used f o r the s t a t i s t i c a l , comparison between c o n t r o l and experimental groups. D i f f e r e n c e s were c o n s i d e r e d s i g n i f i c a n t when p < 0.05. 47 E. METHODOLOGY OF MOLECULAR BIOLOGY 1. MATERIALS B a c t e r i a l c u l t u r e s u p p l i e s and media were purchased from D i f c o ( B u r l i n g t o n , O n t a r i o , Canada). K i o l b a s e DNA sequencing k i t and M o l e c u l a r b i o l o g i c a l s u p p l i e s were from Gibco/BRL ( B u r l i n g t o n , O n t a r i o , Canada). 1 2 5 i ( i mCi/mmol) , 3 2P-dCTP (1 mCi/mmol) and S-dATP were from Amersham ( O a k v i l l e , O n t a r i o , Canada). The GENECLEAN k i t was from BIO 101 Inc. (La J o l l a , CA). Forward and r e v e r s e sequencing primers were from Promega (Madison, WI). The chromosome panel b l o t was from BIOS (New Haven, CT). 2 . BACTERIA STRAIN E . c o l i of s t r a i n Y1090 was used f o r immunoscreening of mouse t e s t i s cDNA l i b r a r y c o n s t r u c t e d with Lambda gt 11 e x p r e s s i o n v e c t o r . S t r a i n Y1089 was used f o r the p r o d u c t i o n of recombinant f u s i o n p r o t e i n . S t r a i n DH5 was used f o r DNA s u b c l o n i n g and sequencing. 3. DNA ISOLATION (The formular of s o l u t i o n s are summarized i n Appendix 7) 48 a. TISSUE DNA PREPARATION High molecular-weight c e l l u l a r DNA from t i s s u e s was e x t r a c t e d by u s i n g the phenol and c h l o r o f o r m method ( M a n i a t i s e t a l . , 1982). B r i e f l y , 1 gm of t i s s u e sample was homogenized i n P o l y t r o n i n 5 ml of Tris-Na-EDTA b u f f e r . Protease K (100 ug/ ml) and 0.5 % SDS were then added t o the t i s s u e homogenate. The mixture was incubated a t 50°C f o r 3 hours. A f t e r i n c u b a t i o n , the d i g e s t e d homogenate was e x t r a c t e d t h r e e times w i t h an equal volume of phenol and once with choloform. The e x t r a c t e d DNA was then p r e c i p i t a t e d by adding one h a l f volume of 7.5 M ammonium a c e t a t e and 2.5 volumes of 95 % e t h a n o l . A f t e r c e n t r i f u g a t i o n , the DNA was d i s s o l v e d i n Tris-EDTA b u f f e r . The c o n c e n t r a t i o n of DNA was determined s p e c t r o p h o t o m e t r i c a l l y a t absorbance of 260 nm. The q u a l i t y of DNA was e v a l u a t e d by agarose g e l e l e c t r o p h o r e s i s . b. BACTERIOPHAGE DNA PREPARATION (The formular of s o l u t i o n s are summarized i n Appendix 8) Bacteriophage lambda DNA was prepared i n sm a l l s c a l e a c c o r d i n g t o the p l a t e l y s a t e method (M a n i a t i s e t a l . , 1982). B r i e f l y , a s i n g l e , w e l l - i s o l a t e d plaque was p i c k e d and t r a n s f e r r e d i n t o 1 ml SM b u f f e r by u s i n g a pasteur p i p e t t e . A f t e r 49 v o r t e x i n g , 100 u l of bacteriophage suspension was mixed with 100 u l of IL_ c o l i c e l l s and incubated a t 37°C f o r 20 minutes. Three ml of molten t o p agarose (0.7 %) which was kept at 50°C was added t o the mixture of bacteriophage and b a c t e r i a c e l l s . I t was spread onto the s u r f a c e of a 85-mm p l a t e c o n t a i n i n g 30 ml of L u r i a - B e r t a n i (LB) medium with 1.5 % agarose. The p l a t e was i n v e r t e d and incubated a t 37°C f o r 12 t o 14 hours u n t i l the plaques covered almost the e n t i r e s u r f a c e of the p l a t e . F i v e ml of SM b u f f e r was added onto the p l a t e and shaken f o r 3 hours a t room temperature t o e l u t e the bacteriophages from the t o p agarose. The washed SM b u f f e r was t r a n s f e r r e d t o a c e n t r i f u g e tube. The b a c t e r i a l d e b r i s was removed by c e n t r i f u g a t i o n a t 8,000 X g f o r 10 minutes a t 4°C. RNase A and DNase I were added t o a f i n a l c o n c e n t r a t i o n of 1 ug/ml t o the supernatant. A f t e r a 30 minute i n c u b a t i o n a t 37°C, an equal volume of a s o l u t i o n c o n t a i n i n g 20 % (w/v) p o l y e t h y l e n e g l y c o l (PEG 8000) and 2 M NaCl i n SM b u f f e r was added t o bacteriophage s o l u t i o n and incubated at 0°C f o r one hour. The p r e c i p i t a t e d bacteriophage p a r t i c l e s were recovered by c e n t r i f u g a t i o n a t 10,000 X g f o r 20 minutes a t 4°C. A f t e r removal of the supernatant, the tube was i n v e r t e d on a paper towel t o d r a i n away remaining l i q u i d . The bacteriophage was resuspended by adding 0.5 ml of SM b u f f e r and shaken v i g o r o u s l y 50 with a v o r t e x meter. The bacteriophage s o l u t i o n was c e n t r i f u g e d a t 8,000 X g f o r 2 minutes a t 4°C. t o remove d e b r i s . The supernatant was t r a n s f e r r e d t o a f r e s h Eppendorf tube, 5 u l each of 10 % SDS and 0.5 M EDTA (pH 8.0) s o l u t i o n s were added. A f t e r i n c u b a t i o n a t 68°C f o r 15 minutes, the supernatant was e x t r a c t e d s e q u e n t i a l l y w i t h phenol, phenol/chloroform (1:1), and then w i t h c h l o r o f o r m . An equal volume of i s o p r o p a n o l was added to the f i n a l aqueous phase t o p r e c i p i t a t e bacteriophage DNA. A f t e r s t o r i n g a t -70°C f o r 20 minutes, the s o l u t i o n was thawed and c e n t r i f u g e d i n an Eppendorf c e n t r i f u g e f o r 15 minutes a t 4°C. The p e l l e t was washed with 70 % e t h a n o l , d r i e d and resuspended i n 50 u l of TE b u f f e r . The e x t r a c t e d bacteriophage DNA was analyzed by r e s t r i c t i o n enzymes d i g e s t i o n f o l l o w e d by agarose g e l e l e c t r o p h o r e s i s . c. PLASMID DNA PREPARATION (M a n i a t i s e t a l . , 1982) (The formular of s o l u t i o n s are summarized i n Appendix 9) One s i n g l e w e l l - i s o l a t e d colony was p i c k e d from the p l a t e and seeded i n 10 ml of LB medium f o r o v e r n i g h t i n c u b a t i o n a t 37°C (with s h a k i n g ) . 10 ml of the w e l l grown p r e c u l t u r e medium was then poured i n t o a 500 ml of LB medium f o l l o w e d by o v e r n i g h t i n c u b a t i o n a t 37°C. The b a c t e r i a l c e l l s were harvested by 51 c e n t r i f u g a t i o n a t 4,000 X g f o r 10 minutes a t 4°C. The c e l l p e l l e t was washed with dH 20 f o l l o w e d by a d d i t i o n a l c e n t r i f u g a t i o n . The b a c t e r i a l p e l l e t was then resuspended i n 10 ml of s o l u t i o n I c o n t a i n i n g 5 mg/ml lysozyme and incubated f o r 5 minutes a t room temperature. 20 ml of f r e s h l y prepared s o l u t i o n I I was added. A f t e r an a d d i t i o n a l 10 minute i n c u b a t i o n i n an i c e bath, 15 ml of i c e - c o l d s o l u t i o n I I I was added and mixed tho r o u g h l y f o r 10 minute on i c e . The p r e c i p i t a t e d p e l l e t was removed by c e n t r i f u g a t i o n a t 4,000 X g f o r 20 minutes a t 4°. The supernatant was t r a n s f e r r e d to a f r e s h tube, the plasmid DNA was p r e c i p i t a t e d by adding two volumes of 95 % ethanol and p l a c e d a t - 7 0 ° C f o r 15 minutes. The DNA p e l l e t was c o l l e c t e d by c e n t r i f u g a t i o n at 10,000 X g f o r 10 minutes a t 4°C and then d r i e d under vacuum. The dry DNA p e l l e t was d i s s o l v e d i n 2 ml of dH 20 and incubated w i t h RNase A a t a c o n c e n t r a t i o n of 200 ug/ml. A f t e r i n c u b a t i o n a t 3 7°C f o r 1 hour, the DNA s o l u t i o n was e x t r a c t e d t w i ce with phenol and once with ch l o r o f o r m . F i n a l l y , i t was p r e c i p i t a t e d by adding 1/10 volume of 7.5 M ammonium a c e t a t e and 2.5 volumes of 95 % e t h a n o l . The q u a l i t y and q u a n t i t y of p u r i f i e d plasmid was determined by r e s t r i c t i o n enzymes d i g e s t i o n f o l l o w e d by agarose g e l e l e c t r o p h o r e s i s . 52 4. RNA ISOLATION (The formular of s o l u t i o n s are summarized i n Appendix 10) An e x t r a c t i o n method i n v o l v i n g the use of a c i d guanidinium t h i o c y a n a t e , phenol and c h l o r o f o r m was used f o r t o t a l RNA i s o l a t i o n (Chomczynski and S a c c h i , 1987). B r i e f l y , 1 gm of f r e s h or f r o z e n t i s s u e ( s t o r e d i n a l i q u i d n i t r o g e n tank) was minced on i c e and homogenized with 10 ml of s o l u t i o n D i n a g l a s s - t e f l o n homogenizer and subsequently t r a n s f e r r e d t o a p o lypropylene tube. A s o l u t i o n c o n t a i n i n g 1 ml of 2 M sodium a c e t a t e , pH 4, 10 ml of phenol (water saturated)., and 2 ml of chloroforra-isoamyl a l c o h o l (49:1) was added t o the homogenate with thorough mixing. The f i n a l suspension was shaken v i g o r o u s l y f o r 10 seconds and c o o l e d on i c e f o r 15 minutes. The suspension was then c e n t r i f u g e d a t 10,000 g f o r 20 minutes at 4°C. A f t e r c e n t r i f u g a t i o n , RNA was p r e s e n t i n the aqueous phase whereas DNA and p r o t e i n s were presen t i n the i n t e r p h a s e and phenol phase. The aqueous phase was t r a n s f e r r e d t o a f r e s h tube f o l l o w e d by mixing with an equal volume of i s o p r o p a n o l , and then p l a c e d a t -20°C f o r 1 hour t o p r e c i p i t a t e RNA. A f t e r c e n t r i f u g a t i o n , the RNA p e l l e t was d i s s o l v e d i n 3 ml of s o l u t i o n D (see Appendix 10) and p r e c i p i t a t e d with 1 volume of i s o p r o p a n o l . A f t e r washing with 75 % e t h a n o l , the RNA p e l l e t was vacuum d r i e d and then d i s s o l v e d i n 53 0.5 ml of 0.5 % SDS. The s o l u t i o n was then incubated a t 65°C f o r 10 minutes. The c o n c e n t r a t i o n and q u a l i t y of e x t r a c t e d RNA were determined s p e c t r o p h o t o m e t r i c a l l y by absorbance a t 260 nm and 280 nm and agarose g e l e l e c t r o p h o r e s i s , r e s p e c t i v e l y . 5. NICK TRANSLATION OF DNA (M a n i a t i s e t a l . , 1982) (The formular o f s o l u t i o n s are summarized i n Appendix 11) The cDNA i n s e r t was e x c i s e d from a v e c t o r by r e s t r i c t i o n enzyme d i g e s t i o n . The fragment was p u r i f i e d on an agarose g e l by u s i n g a GENECLEAN k i t . The p u r i f i e d DNA fragment (0.5 ug) was mixed with a s o l u t i o n c o n t a i n i n g 6 u l of d e o x y r i b o n u c l e o s i d e t r i p h o s p h a t e (dNTP) mixture, 2 u l of 10 X n i c k t r a n s l a t i o n b u f f e r , 8 u l of 3 2 P l a b e l e d a-dCTP and 4 u l of dH 20. A f t e r the a d d i t i o n of 1 u l of 100 ng/ml DNase I and 10 u n i t s of EL. c o l i DNA polymerase I, the mixture was f i r s t incubated a t 37°C f o r 10 minutes and then a t 15°C f o r 2 hours. During i n c u b a t i o n , 1 ml of Sephadex G50 was packed i n a 1 ml s y r i n g e and was washed wit h STE b u f f e r . The packed column was c e n t r i f u g e d a t 1600 X g f o r 4 minutes f o r washing and s e p a r a t i n g the f r e e n u c l e o t i d e . A f t e r i n c u b a t i o n , 2 u l of 10 % SDS and 2 u l of 0.5 M EDTA were added to the mixture t o stop the r e a c t i o n . F o l l o w i n g the a d d i t i o n of 5 u l 54 of y e a s t tRNA (10 mg/ml) and 180 u l of STE b u f f e r , the mixture i n a f i n a l volume of 200 u l was a p p l i e d t o a Sephadex G-50 column which was then c e n t r i f u g e d . The e l u t e d DNA s o l u t i o n was c o l l e c t e d i n a 1.5 ml Eppendorf tube. The r a d i o a c t i v i t y of l a b e l e d DNA was d e t e c t e d by a beta s c i n t i l l a t i o n counter. The s p e c i f i c a c t i v i t y was presented i n cpm/ug DNA. 6. SOUTHERN BLOT HYBRIDIZATION (The formular of s o l u t i o n s are summarized i n Appendix 12) a. SOUTHERN TRANSFER Southern b l o t analyses of the DNA were performed a c c o r d i n g to the standard procedures (Southern, 1975). A f t e r s i z e -f r a c t i o n a t i o n i n agarose g e l e l e c t r o p h o r e s i s , the DNA on the g e l was denatured i n a s o l u t i o n c o n t a i n i n g 0.5 M NaOH and 1.5 M NaCl f o r 1 hour and then n e u t r a l i z e d i n 1 M T r i s - H C l pH 8.0 c o n t a i n i n g 1.5 M NaCl f o r 1 hour w i t h smooth shaking. Subsequently, the DNA was t r a n s f e r r e d by pressure to a nylon f i l t e r w ith a t r a n s f e r b u f f e r (20 X SSC) and then immobilized by baking a t 80°C f o r 2 hours or by UV l i g h t (Khandjian, 1987) f o r 2 minutes. The dry f i l t e r s were s t o r e d i n a s e a l e d bag a t room temperature. b . HYBRIDIZATION 55 The b l o t t e d f i l t e r was p r e h y b r i d i z e d i n a h y b r i d i z a t i o n s o l u t i o n a t 65°C f o r 30 minutes and h y b r i d i z e d i n the same s o l u t i o n w i t h a 3 2 P - l a b e l e d DNA probe o v e r n i g h t a t 6 5°C. A f t e r h y b r i d i z a t i o n , the h y b r i d i z e d f i l t e r was washed twice i n 2 X SSC c o n t a i n i n g 0.5 % SDS a t room temperature f o r 15 minutes and then once a t 50°C f o r 30 minutes. The b l o t was then a i r d r i e d and autoradiographed by u s i n g i n t e n s i f y i n g screens a t -70°C. 7. NORTHERN BLOT HYBRIDIZATION T o t a l RNA from t i s s u e s was i s o l a t e d , denatured by g l y o x a l a t i o n and s i z e - f r a c t i o n a t e d i n 1.2 % agarose g e l e l e c t r o p h o r e s i s (Varmus e t a l . , 1981, M a n i a t i s e t a l . , 1982). The RNA was t r a n s f e r r e d by pressure t o nylon f i l t e r s and h y b r i d i z e d with a P - l a b e l e d DNA probe as p r e v i o u s l y d e s c r i b e d . 8. IMMUNOSCREENING OF LAMBDA GT 11 CDNA LIBRARY (The formular of s o l u t i o n s are summarized i n Appendix 13) a. AMPLIFICATION OF cDNA LIBRARY The mouse t e s t i s cDNA l i b r a r y was obtained from Dr. Y.F. Lau's l a b a t the U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o , and was c o n s t r u c t e d i n a bacteriophage e x p r e s s i o n v e c t o r , lambda g t l l by u s i n g p u r i f i e d p o l y ( A ) + mRNA from the testes of 3-month o l d 56 BALB/C mice. The mouse t e s t i s cDNA l i b r a r y was i n SM b u f f e r and the t i t r e of the l i b r a r y was determined by a s e r i a l d i l u t i o n of the l i b r a r y which was p l a t e d on LB p l a t e s as d e s c r i b e d p r e v i o u s l y . For the a m p l i f i c a t i o n of the l i b r a r y , 10 J recombinant phages were p l a t e d on a 150 mm LB p l a t e . A f t e r i n c u b a t i o n o v e r n i g h t a t 37°C, 10 ml of SM b u f f e r was added t o resuspend the phage by shaking a t room temperature f o r 2 hours. The t i t r e of resuspended cDNA l i b r a r y was redetermined p r i o r t o use. b. PREPARATION OF POLYCLONAL ANTISERA F i v e ml of r a b b i t anti-MSA-63 s e r a were p r e c i p i t a t e d with 50 % (w/v) ammonium s u l f a t e . A f t e r c e n t r i f u g a t i o n , the p r o t e i n p e l l e t was d i s s o l v e d i n 5 ml of TBS and d i a l y z e d a g a i n s t TBS ov e r n i g h t . The d i a l y z e d antibody s o l u t i o n was used f o r immunoscreening of the cDNA l i b r a r y . c. PROCEDURES OF IMMUNOSCREENING OF MOUSE TESTIS CDNA LIBRARY Immunoscreening was performed a c c o r d i n g t o the i n s t r u c t i o n of the C l o n t e c h Lambda g t 11 Immunoscreening K i t with some m o d i f i c a t i o n s . The standard p r o t o c o l of immunoscreening i s presented i n Fi g u r e 1. B r i e f l y , a s i n g l e , i s o l a t e d colony of E. c o i i Y1090 was p i c k e d and grown t o s a t u r a t i o n i n LB br o t h 57 c o n t a i n i n g 0.2 % maltose a t 37°C wi t h good a e r a t i o n . 0.2 ml of the Y1090 c u l t u r e and 0.1 ml of cDNA l i b r a r y c o n t a i n i n g 0.5 X 10 5 recombinant phage were mixed and incubated a t 37°C f o r 15 minutes. A f t e r the a d d i t i o n of 7.5 ml of molten LB s o f t agar, the c u l t u r e was mixed and poured onto a 150 mm LB p l a t e and then incubated a t 42°C f o r 3.5 hours. The cDNA l i b r a r y was poured onto twelve 150 mm LB p l a t e s f o r immunoscreening. A f t e r i n c u b a t i o n , the p l a t e s were t r a n s f e r r e d t o a 37°C i n c u b a t o r and a dry n i t r o c e l l u l o s e f i l t e r which was p r e v i o u s l y s a t u r a t e d with 10 mM IPTG was o v e r l a y e d t o each p l a t e and incubated f o r 3.5 hours. The f i l t e r s were then removed from the p l a t e s and incubated i n T r i s b u f f e r e d s a l i n e w i t h Tween 20 c o n t a i n i n g 20 % f e t a l c a l f serum f o r 30 minutes a t room temperature f o r b l o c k i n g . Rabbit anti-MSA-63 s e r a a t a c o n c e n t r a t i o n of 2 u l / 10 ml TBST was added t o f i l t e r s and incubated o v e r n i g h t a t room temperature. A f t e r three washes of TBST, these f i l t e r s were incubated w i t h b i o t i n y l a t e d goat a n t i - r a b b i t IgG antibody a t a c o n c e n t r a t i o n of 20 u l / 10 ml TBST f o r 30 minutes. The f i l t e r s were washed by TBST and incubated w i t h a v i d i n - b i o t i n y l a t e d h o r s e r a d i s h peroxidase complex f o r 30 minutes. The f i l t e r s were then washed by TBS (without Tween 20) and incubated i n a peroxidase s u b s t r a t e s o l u t i o n f o r 30 minutes t o develop the c o l o r . The p o s i t i v e plaques were 58 F i g u r e 1. P r o t o c o l of immunoscreening of cDNA l i b r a r y with antibody probe. ( M o d i f i e d from i n s t r u c t i o n of C l o n t e c h immunoscreening K i t ) 59 Plate Agll 1 dunes on K. c n l i YIO'X) and incubate al 42"C" lor J ! ' : hours I'lia^c plaque Overlay wiih IPTG-saluraled tiller t lur i'/i hours Remove tiller and probe vviih primary antibody I'rohe lor hound antibody followed by inmiunoperoudase staining Positive signals | 60 i d e n t i f i e d and p i c k e d f o r a second s c r e e n i n g by the same method. d. IDENTIFICATION OF POSITIVE CLONES The DNA of p o s i t i v e recombinant phage c l o n e s was e x t r a c t e d a c c o r d i n g t o the method d e s c r i b e d i n s e c t i o n E.3.b., and d i g e s t e d by EcoRI r e s t r i c t i o n enzyme. A f t e r the agarose g e l e l e c t r o p h o r e s i s , the DNA was t r a n s f e r r e d t o a n i t r o c e l l u l o s e f i l t e r paper by Southern b l o t t i n g and h y b r i d i z e d by u s i n g one of the cDNA i n s e r t s as a probe. Two cDNA i n s e r t s were probes f o r the h y b r i d i z a t i o n . The l o n g e s t cDNA i n s e r t which c r o s s - r e a c t e d with the others was s e l e c t e d f o r s u b c l o n i n g and f o r f u r t h e r a n a l y s i s . 9 . SUBCLONING OF DNA INSERTS a. PREPARATION OF RECOMBINANT PLASMID DNA FROM RECOMBINANT BACTERIOPHAGE The p o s i t i v e c l o n e s which were i d e n t i f i e d from the bacteriophage cDNA l i b r a r y were subcloned i n t o a pUC18 plasmid v e c t o r f o r the f u r t h e r r e s t r i c t i o n a n a l y s i s and DNA sequencing. The DNA i n s e r t from the recombinant bacteriophage DNA was completely d i g e s t e d by EcoR I and recovered from the agarose g e l us i n g the GENECLEAN k i t . The i s o l a t e d DNA i n s e r t was then l i g a t e d 61 with the pUC18 v e c t o r which was d i g e s t e d by EcoRI. The DNA r a t i o of i n s e r t e d DNA t o plasmid was about 1 t o 10. A f t e r the completion o f l i g a t i o n , the recombinant plasmid was ready t o be t r a n s f e r r e d t o the b a c t e r i a l h o s t . b. L . ' c o i i TRANSFORMATION To prepare competent c e l l s f o r t r a n s f o r m a t i o n , a s i n g l e c olony of host s t r a i n DH5 was incubated w i t h 10 ml of LB a t 37°C o v e r n i g h t with c o n s t a n t shaking. One ml of the c e l l c u l t u r e was added t o 50 ml of LB and incubated f o r 2 t o 3 hours u n t i l the c e l l d e n s i t y reached an absorbance of 0.6 a t 550 nm. The c u l t u r e was c h i l l e d on i c e f o r 5 minutes and ha r v e s t e d by c e n t r i f u g a t i o n f o r 5 minutes a t 2,500 X g a t 4°C. The c e l l p e l l e t was resuspended i n 18 ml of 100 mM C a C l 2 and kept f o r 15 minutes on i c e . A f t e r a d d i t i o n a l c e n t r i f u g a t i o n , the c e l l p e l l e t was resuspended i n 2 ml of 100 mM C a C l 2 and kept f o r 60 minutes a t 4°C. A f t e r the above p r e p a r a t i o n , the c e l l s were ready f o r DNA t r a n s f o r m a t i o n . In a s t e r i l e Eppendorf tube, 10 u l of l i g a t e d recombinant plasmid was mixed with 200 u l of competent c e l l s f o r 3 0 minutes a t 4°C. The Eppendorf tube c o n t a i n i n g the mixture was t r a n s f e r r e d t o a 42°C water bath f o r 2 minutes t o heat-shock the c e l l s and then t r a n s f e r r e d t o a 37°C water bath f o r 1 hour shaking i n the presence of 1 ml of LB medium to al l o w e x p r e s s i o n of the a n t i b i o t i c r e s i s t a n c e gene. One hundred u l of the c e l l mixture was spread over the s u r f a c e of an L B / a g a r / a m p i c i l l i n p l a t e (85 mm) f o r o v e r n i g h t c u l t u r e . The p o s i t i v e colony was s e l e c t e d by colony h y b r i d i z a t i o n w i t h a probe of 3 2 P l a b e l e d i n s e r t DNA. 10. DNA SEQUENCING The dideoxy sequencing method by means of denatured plasmid templates was used f o r the DNA sequence a n a l y s i s ( H a t t o r i and Sakaki, 1986). a. PREPARATION AND ALKALINE DENATURATION OF PLASMID DNA The EcoRI d i g e s t e d cDNA i n s e r t was subcloned t o the plasmid pUC18 and t h i s recombinant plasmid was e x t r a c t e d a c c o r d i n g t o the method p r e v i o u s l y d e s c r i b e d . Eighteen u l of p u r i f i e d plasmid DNA s o l u t i o n (0.2 ug/ul) was mixed with 2 u l of 2 N NaOH and kept a t room temperature f o r 5 minutes. Then, 8 u l of 5 M ammonium a c e t a t e (pH 7.4) was added and the denatured DNA was p r e c i p i t a t e d by the a d d i t i o n of 100 u l of ethanol and p l a c e d a t -70°C f o r 5 minutes. The p r e c i p i t a t e was harvested by c e n t r i f u g a t i o n a t 12,000 rpm f o r 5 minutes, r i n s e d once with 70 % et h a n o l , and 63 d r i e d under vacuum- The denatured plasmid DNA p e l l e t was d i s s o l v e d i n 6.5 u l of dH 20 bef o r e use. b. Enzyme Re a c t i o n f o r Sequencing Enzyme r e a c t i o n s f o r sequencing i n c l u d i n g template-primer a n n e a l i n g and l a b e l i n g - e x t e n s i o n r e a c t i o n s were d e s c r i b e d i n the i n s t r u c t i o n manual of the K i l o B a s e Sequencing System (BRL). B r i e f l y , the mixture of 6.5 u l of denature plasmid (5 ug), 1.5 u l of primer (0.5 pmol) and 2 u l of K i l o B a s e Sequencing B u f f e r (10 X) was heated t o 65°C f o r 10 minutes. The mixture was allowed t o c o o l s l o w l y t o room temperature (about 30 minutes). A f t e r the temperature e q u i l i b r a t i o n , 2 u l of d i l u t e d (IX) K i l o B a s e E x t e n s i o n Mix, 1 u l of 1 M d i t h i o t h r e i t o l , 1 u l 3 5 S - l a b e l e d dATP and water were added t o the mixture of denatured plasmid. A f t e r i n c u b a t i o n f o r 5 minutes a t 37°C, 1 u l of the l a r g e fragment of DNA polymerase I (1.5 u n i t s / u l ) was added with an a d d i t i o n a l i n c u b a t i o n a t 37°C f o r 5 minutes. 3.5 u l of r e a c t i o n mixture was then t r a n s f e r r e d r e s p e c t i v e l y t o each of the f o u r tubes c o n t a i n i n g K i l o b a s e Termination Mixes-A, -C, -G, and -T. A f t e r an i n c u b a t i o n p e r i o d of 5 minutes a t 37°C, the r e a c t i o n was stopped by adding 4 u l of dideoxy stop b u f f e r . F i n a l l y , the r e a c t i o n mixture was denatured by h e a t i n g the tubes at 80°C f o r 5 64 minutes f o l l o w e d by a r a p i d t r a n s f e r t o wet i c e . C. SEQUENCING The sequencing g e l (38 X 50 cm, 0.4 mm) was prepared a c c o r d i n g t o the method d e s c r i b e d i n the i n s t r u c t i o n manual of Sequi-Gen N u c l e i c A c i d Sequencing C e l l (BIO-RAD). Three u l of each r e a c t i o n s o l u t i o n was a p p l i e d t o one lane of the sequencing g e l . A f t e r the g e l had been run a t a con s t a n t v o l t a g e o f 2000 V f o r 2.5 hours, the g e l was f i x e d i n a s o l u t i o n c o n t a i n i n g 10 % methanol and 10 % a c e t i c a c i d f o r 15 minutes. Then, the f i x e d g e l was d r i e d a t 80°C f o r 1 hour by u s i n g a g e l d r y e r under vacuum. Fi n a l l y . , the d r i e d g e l was p l a c e d i n a c a s s e t t e w i t h X-ray f i l m f o r autoradiography. The r e s u l t of the sequence was analyzed by u s i n g PC-GENE computer program. 11. PRODUCTION OF RECOMBINANT FUSION PROTEIN Crude l y s a t e s of Ej. c o l i s t r a i n Y1089 which expressed MSA-63 f u s i o n p r o t e i n were prepared a c c o r d i n g t o the p u b l i s h e d procedures (Snyder e t a l , 1987). a. LYSOGENIC STRAIN PREPARATION The s a t u r a t e d c u l t u r e of Y1089 c e l l s i n a LB medium 65 c o n t a i n i n g 0.2 % maltose and 50 ug/ml of a m p i c l l i n was i n f e c t e d w i t h MSA-63 recombinant phage c l o n e s u s i n g an m . o . i . ( m u l t i p l i c i t y of i n f e c t i o n ) equal to 10 f o r 20 minutes a t room temperature. Two hundred c e l l s were p l a t e d per LB p l a t e and incubated o v e r n i g h t a t 30°C. The i n d i v i d u a l c o l o n i e s were p i c k e d w i t h a t o o t h p i c k and t e s t e d f o r growth a t 42°C and 30°C. The lysogens s e l e c t i o n was based on t h e i r growth a t 30°C, but no growth a t 42°C. b. PREPARATION OF FUSION PROTEIN Lysogens were grown i n LB medium with v i g o r o u s shaking a t 30°C u n t i l an absorbance of 0.4 a t 600 nm. Lysogens were induced by s h i f t i n g the temperature to 44°C f o r 15 minutes with v i g o r o u s shaking. IPTG was then added t o the c u l t u r e t o a f i n a l c o n c e n t r a t i o n of 10 mM. A f t e r i n c u b a t i o n a t 37°C f o r 20 minutes, the c e l l s were ha r v e s t e d as q u i c k l y as p o s s i b l e by c e n t r i f u g a t i o n at 10,000g f o r 30 seconds and the c e l l p e l l e t was f r o z e n immediately a t -70°C. For SDS-PAGE, SDS sample b u f f e r was added j u s t p r i o r t o f r e e z i n g . For the radioimmunosorbent i n h i b i t i o n assay, the harvested c e l l p e l l e t was s o n i c a t e d and incubated immediately with i 1 2 5 l a b e l e d HS-63 c o n t a i n i n g 2 mM PMSF. For the i s o l a t i o n of MSA-63 recombinant f u s i o n p r o t e i n , the LB medium c o n t a i n i n g l y s e d lysogens was c o n c e n t r a t e d t o a s m a l l 66 volume by u s i n g Amicon pressure c e l l and a p p l i e d t o a Sephacryl S-300 column. The e l u e n t which had a molecular s i z e of g r e a t e r than 100 Kd was c o l l e c t e d and used t o immunize r a b b i t s f o r the p r o d u c t i o n of p o l y c l o n a l a n t i s e r a . 12. SCREENING OF cDNA LIBRARY BY DNA PROBE The mouse t e s t i s cDNA l i b r a r y was p l a t e d on a 15 mm LB agar p l a t e a t a d e n s i t y of 5 x 10 4 plaques and grown o v e r n i g h t a t 37°C. The p l a t e was then c h i l l e d a t 4°C f o r 1 hour t o a l l o w the top agar l a y e r t o harden. A dry nylon f i l t e r was p l a c e d onto the s u r f a c e of the t o p agar f o r 1 minute at room temperature t o absorb the recombinant plaques. A f t e r removing from the p l a t e , the f i l t e r was p l a c e d i n a d e n a t u r i n g s o l u t i o n (1.5 M NaCl, 0.5 M NaOH) f o r 1 minute, and then t r a n s f e r r e d i n t o ' n e u t r a l i z i n g s o l u t i o n (1.5 M NaCl, 0.5 M T r i s - H C l pH 8.0) f o r 5 minutes, and a i r d r i e d . A f t e r the f i l t e r was d r i e d , DNA was f i x e d t o the f i l t e r by baking or UV l i g h t f o r the h y b r i d i z a t i o n as d e s c r i b e d p r e v i o u s l y . 67 F. IMMUNOHISTOCHEMISTRY For the p r e p a r a t i o n o f f r o z e n t e s t i c u l a r s e c t i o n s , t e s t e s were removed from male mice of d i f f e r e n t ages, (from b i r t h t o matur i t y ) and then f r o z e n immediately i n l i q u i d n i t r o g e n . Subsequently, they were p l a c e d i n an embedding medium ( T i s s u e TeKII, O.C.T. Compound No. 4583, Lab-Tek) a t -60°C. S e c t i o n s of 10 urn t h i c k n e s s were c u t and then p l a c e d on s l i d e s , a i r d r i e d , f i x e d i n 100 % methanol, and s t o r e d a t -20°C. The i n d i r e c t immunofluorescent assay was used f o r immunohistological s t u d i e s of t i s s u e s e c t i o n s a c c o r d i n g t o the procedures d e s c r i b e d p r e v i o u s l y (Lee and Wong, 1986b). 68 I I I . RESULTS P a r t I . CHARACTERIZATION OF HS-63 MONOCLONAL ANTIBODY AND EVALUATION OF ITS CORRESPONDING SPERM ANTIGENS A^ . LOCALIZATION OF SA-6 3 ANTIGEN IN SPERM By u s i n g i n d i r e c t immunofluorescent assay, HS-63 monoclonal antibody was shown t o r e a c t w i t h the acrosome of sperm among d i f f e r e n t mammalian s p e c i e s . The i n d i r e c t immunofluorescent s t a i n i n g p a t t e r n s of HS-6 3 to mouse and human sperm are presented i n F i g u r e 2. Both a i r - d r i e d and methanol-fixed acrosome-intact sperm were s t a i n e d p o s i t i v e l y i n the acrosomal r e g i o n upon i n c u b a t i o n w i t h t h i s antibody. R e a c t i v i t y of HS-6 3 t o l i v e sperm was a l s o examined by d i r e c t immunofluorescent a n a l y s i s . Percent immunofluorescent s t a i n i n g of f r e s h l y prepared l i v e mouse sperm from cauda e p i d i d y m i d i s and vas deferens were found t o i n c r e a s e d r a m a t i c a l l y with time upon 30 minutes i n c u b a t i o n with F I T C - l a b e l e d HS-63. However, F I T C - l a b e l e d HS-63 was found t o have l i t t l e or n o n s t a i n i n g with f r e s h l y prepared l i v e sperm which were f i x e d w i t h g l u t a r a l d e h y d e . 69 F i g u r e 2. I n d i r e c t immunofluorescent s t a i n i n g of mouse (A, B) and human (C, D) sperm by u s i n g HS-63 monoclonal antibody. Sperm under UV l i g h t are presented i n (A, C) and sperm under v i s i b l e l i g h t are gi v e n i n (B, D) (X 400). 70 71 On the other hand, upon 1 hour i n c u b a t i o n of l i v e sperm i n the BWW medium f o r c a p a c i t a t i o n , g l u t a l d e h y d e - f i x e d c a p a c i t a t e d sperm were s t a i n e d p o s i t i v e l y with F I T C - l a b e l e d HS-63. The immunofluorescent s t a i n i n g p a t t e r n of c a p a c i t a t e d mouse sperm upon i n c u b a t i o n with HS-63 i s presented i n F i g u r e 3. When f r e s h l y prepared sperm were washed wi t h 0.5 M KC1 or 0.1 % T r i t o n X-100, they were r e a d i l y s t a i n e d w i t h the F I T C - l a b e l e d HS-63.(Menge e t a l . , 1987). F o l l o w i n g i n c u b a t i o n of sperm wi t h c a l c i u m ionophore A-23187 to induce acrosome r e a c t i o n , the acrosome-reacted sperm c o u l d no longer be s t a i n e d w i t h FITC-l a b e l e d HS-63. B^ EPITOPE EVALUATION OF HS-63 By u s i n g HRP-labeled HS-63 as a probe, the immunoactivity of the a n t i g e n i n mouse t e s t i s homogenate was not s i g n i f i c a n t l y decreased f o l l o w i n g the treatment w i t h T r i t o n X-100 or NP-40. However, no HS-63 immunoactivity was d e t e c t e d when the t e s t i s homogenate was prepared i n the presence of SDS. 72 F i g u r e 3. Immunofluorescent s t a i n i n g of c a p a c i t a t e d mouse sperm by u s i n g F I T C - l a b e l e d HS-63. Sperm under UV l i g h t are presented i n ( A ) and sperm under v i s i b l e l i g h t are g i v e n i n (B) (X 400). 73 74 Treatments w i t h p r o t e o l y t i c enzymes, such as t r y p s i n and pronase, were shown t o completely a b o l i s h the immunoactivity of the a n t i g e n i n mouse t e s t i s homogenate. However, i n c u b a t i o n s o f t e s t i s homogenate with sodium p e r i o d a t e and neuraminidase which e i t h e r m o d i f i e s or removes carbohydrate moiety o f g l y c o p r o t e i n s have l i t t l e e f f e c t t o the .immunoactivity of the a n t i g e n . ( L . EVALUATION OF HS-63 ON. SPERM FUNCTION ( L i u e t a l . , 1989) 1. IN VITRO FERTILIZATION OF MOUSE OOCYTES Because of the c r o s s - r e a c t i v i t y o f HS-63 to sperm of v a r i o u s mammalian s p e c i e s , the i n h i b i t o r y e f f e c t of HS-63 on f e r t i l i z a t i o n was e v a l u a t e d by u s i n g mouse i n v i t r o f e r t i l i z a t i o n experiment. The h e a t - i n a c t i v a t e d a s c i t e s f l u i d c o n t a i n i n g HS-63 monoclonal antibody (antibody t i t r e > 1:100,000) showed 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 f e r t i l i z a t i o n of mouse oocytes a t a d i l u t i o n of 1:10 ( F e r t i l i z a t i o n r a t e of 24.6 % vs. 57.7 % f o r the c o n t r o l , P < 0.001). The r e s u l t s of t h i s study are presented i n Table 1. I t was observed d u r i n g t h i s experiment t h a t HS-63 n e i t h e r had an e f f e c t on sperm m o t i l i t y nor caused any sperm a g g l u t i n a t i o n . 75 T a b l e 1. I n h i b i t o r y e f f e c t o f HS-63 monoclonal a n t i b o d y on i n v i t r o f e r t i l i z a t i o n of mouse oocytes. A n t i b o d i e s Number of Ova examined Ova f e r t i l i z e d F e r t i l i z a t i o n P ° Added assays (No.) (No.) r a t e (%) v a l u e HS-63 3 2 57 14 24.6 C o n t r o l b 2 45 26 57.7 <0.001 a T h e t i t r e of HS-63 was determined by i n d i r e c t immunofluorescent assay t o be 1:100,000 i n a s c i t e s f l u i d . ^ A s c i t e s f l u i d d e r i v e d from h y b r i d c e l l s s e c r e t i n g u n r e l a t e d a n t i b o d i e s was used as t h e c o n t r o l . cCompared wi t h the c o n t r o l group. To e v a l u a t e the dose-dependent i n h i b i t o r y e f f e c t of HS-63 on f e r t i l i z a t i o n of mouse oocytes i n v i t r o , p u r i f i e d HS-63 monoclonal antibody of d i f f e r e n t c o n c e n t r a t i o n s was added t o sperm-egg i n c u b a t i o n mixture. When the c o n c e n t r a t i o n of HS-63 i n the i n c u b a t i o n mixture i s high e r than 0.2 mg/ml, the f e r t i l i z a t i o n i n h i b i t i o n by the antibody was found t o be s i g n i f i c a n t (P < 0.05). The i n h i b i t o r y e f f e c t of HS-63 on f e r t i l i z a t i o n was found t o decrease g r a d u a l l y w i t h d e c r e a s i n g antibody c o n c e n t r a t i o n (from 0.2 t o 0.0016 mg/ml). At the antibody c o n c e n t r a t i o n lower than 0.04 mg/ml, no 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 was found on f e r t i l i z a t i o n ( f e r t i l i z a t i o n r a t e of 45.6 % v s . 52.9 % f o r the c o n t r o l group). The r e s u l t s of t h i s dose-dependent assay are presented i n Table 2. 77 Table 2. I n h i b i t o r y e f f e c t of p u r i f i e d HS-63 on in v i t r o f e r t i l i z a t i o n of mouse oocytes. C o n c e n t r a t i o n T i t e r 3 Ova examined Ova f e r t i l i z e d F e r t i l i z a t i o n P V a l u e b (rag/ml) (No. ) (No. ) r a t e (%) 1 1:200,000 19 4 21 <0.05 0.2 1: 40,000 55 13 23.6 <0.05 0.04 1: 8,000 46 2.1 45.6 NS 0.008 1: 2,000 37 15 40.5 NS 0.0016 54 28 51.8 NS 0 ( c o n t r o l ) — 17 9 52.9 a T h e t i t r e of antibody was determined by i n d i r e c t immunofluorescent assay, kCompared w i t h the c o n t r o l group. NS: not s i g n i f i c a n t 2. HUMAN SPERM PENETRATION ASSAY In human sperm p e n e t r a t i o n assay, the dose-dependent i n h i b i t o r y e f f e c t o f HS-6 3 on human sperm p e n e t r a t i o n of zona-f r e e hamster eggs was ev a l u a t e d . In the presence of 1 mg/ml of HS-63, the p e n e t r a t i o n r a t e was s i g n i f i c a n t l y reduced as compared t o t h a t of the c o n t r o l . The presence of t h i s antibody had not on l y reduced the mean number of swollen sperm head per egg (1.3 ± 1.2 v s . 4.2 ± 2 . 1 f o r c o n t r o l group, P < 0.0001) but a l s o t h a t of the sperm att a c h e d t o each egg (6.1 ± 6.3 vs. 63.3 ± 21.6 f o r c o n t r o l group, P < 0.0001). The r e s u l t s of t h i s study are presented i n Table 3. S i m i l a r t o mouse i n v i t r o f e r t i l i z a t i o n , the p e n e t r a t i o n r a t e and sperm attachment i n t h i s assay were found t o decrease g r a d u a l l y with i n c r e a s i n g antibody c o n c e n t r a t i o n . L i t t l e or no e f f e c t was observed a t an antibody c o n c e n t r a t i o n lower than 0.04 mg/ml. 79 T a b l e 3. I n h i b i t o r y e f f e c t of p u r i f i e d HS-63 on human sperm p e n e t r a t i o n of zona-f r e e hamster ova. Antibody Ova F e r t i l i z a t i o n Swollen sperm a A t t a c h e d P C o n c e n t r a t i o n examined r a t e head sperm val u e * 3 (mg/ml) (No.) (%) (Avg. No./Egg) (Avg. No./Egg) 1 19 73.6 1.3 ± 1.2 6.1 ± 6.3 <0.0001 0.2 18 100 2.7 ± 1-1 16.1 ± 10.5 <0.0001 0.04 16 100 4.8 ± 2.2 70.5 ± 32.5 NS 0.008 14 100 3.6 ± 0.8 79.0 ± 37.0 NS 0 ( c o n t r o l ) 16 100 . 4.2 ± 2.1 63.3 ± 21.6 Average number of swollen sperm head per egg = Sperm p e n e t r a t i o n r a t e , 'compared wi t h the c o n t r o l group. NS: not s i g n i f i c a n t 3. THE EFFECT OF HS-63 ON INDUCED ACROSOME REACTION The a c i d s o l u b i l i z e d mouse zona p r o t e i n i s known t o induce sperm acrosome r e a c t i o n i n a time- and dose-dependent manner ( S a l i n g e t a l . , 1986). The e f f e c t of HS-63 monoclonal antibody on the zona-induced acrosome r e a c t i o n was e v a l u a t e d . I t was c l e a r l y demonstrated t h a t the percentage of acrosome-reacted mouse sperm was s i g n i f i c a n t l y decreased i n the presence of HS-63 as compared t o t h a t of the c o n t r o l . In a t y p i c a l experiment, upon 30 minutes i n c u b a t i o n with HS-63 (1:10 d i l u t i o n of h e a t e d - i n a c t i v a t e d a s c i t e s f l u i d ) and s o l u b i l i z e d zona, the percentage of acrosome-r e a c t e d sperm was 10.7 % as compared to 38.8 % i n the c o n t r o l . 4. IN VIVO MATING EXPERIMENT BY PASSIVE IMMUNIZATION IN MOUSE For comparative purposes, i n v i v o mating experiments were a l s o performed t o e v a l u a t e the a n t i f e r t i l i t y e f f e c t of HS-63 i n mice. The h e a t - i n a c t i v a t e d a s c i t e s f l u i d c o n t a i n i n g h i g h t i t r e s of HS-63 monoclonal antibody (>1:100,000) was i n j e c t e d i n t r a p e r i t o n e a l l y i n t o female mice t o study t h e i r a n t i f e r t i l i t y e f f e c t s . U n r e l a t e d a s c i t e s f l u i d was used as the ne g a t i v e c o n t r o l . In t h i s experiment, a s c i t e s f l u i d s c o n t a i n i n g MS-204 or 81 MS-207 monoclonal antisperm antibody served as the p o s i t i v e c o n t r o l s . Previous s t u d i e s i n our l a b o r a t o r y (Lee and Wong, 1986a) have shown t h a t these two monoclonal a n t i b o d i e s were mouse sperm s p e c i f i c and capable of i n h i b i t i n g both i n v i t r o and i n v i v o f e r t i l i z a t i o n . R e s u l t s are t a b u l a t e d i n percentages of two-c e l l embryos recovered i n a gi v e n mating experiment as the apparent f e r t i l i z a t i o n r a t e s (Table 4 ) . S t a t i s t i c a l l y , the i n h i b i t o r y e f f e c t of HS-63 on the i n v i v o f e r t i l i z a t i o n of p a s s i v e l y immunized mice was not s i g n i f i c a n t (Percentage of two-c e l l embryos being 87.2 ± 17.3 vs. 97.0+0.05 f o r the c o n t r o l ) . As determined by the i n d i r e c t immunofluorescent assay, the t i t r e of HS-63 i n the p a s s i v e l y immunized mice was found t o be about 1:50,000 to 1:100,000 i n the mouse s e r a , but o n l y 1:2,500 i n the f l u i d of o v i d u c t . In the p o s i t i v e c o n t r o l s , the a s c i t e s f l u i d s of MS-204 and MS-207 were shown to i n h i b i t the mouse i n v i v o f e r t i l i z a t i o n s i g n i f i c a n t l y as judged from the r e l a t i v e l y low recovery r a t e s of t w o - c e l l embryos as compared t o the negative c o n t r o l (48.5 ± 37.5 and 59.5 + 31.6, r e s p e c t i v e l y vs. 97.0 ± 0 .05) 82 T a b l e 4. E f f e c t s of a n t i b o d i e s on the i n v i v o f e r t i l i z a t i o n of mouse o o c y t e s f o l l o w i n g p a s s i v e immunization and mating experiments A n t i b o d i e s 2 No. of Mice Percentage of two c e l l b P v a l u e 0 embryos (%) c o n t r o l 12 97 ± 5.9 HS-63 8 87 ± 17.3 R a b b i t a n t i -MSA-63 8 6 7 + 2 9 . 4 <0.05 MS-204 10 48 + 37.5 <0.001 MS-207 9 59 + 31.6 <0.01 a A s c i t e s f l u i d s and a n t i s e r a were h e a t - i n a c t i v a t e d a t 56°C f o r 30 minutes p r i o r t o i n t r a p e r i t o n e a l i n j e c t i o n . k P e r c e n t a g e o f two c e l l embryos = number o f two c e l l embryos / t o t a l ova r e c o v e r e d . cCompared w i t h the c o n t r o l group. NS: not s i g n i f i c a n t P a r t I I . PURIFICATION AND CHARACTERIZATION OF MSA-63 AND EVALUATION OF ANTIFERTILITY EFFECTS BY ITS ISOIMMUNE SERA Aj, PURIFICATION OF HS-63 SPECIFIC ANTIGEN FROM MOUSE TESTES  (MSA-63) ( L i u e t a l . , 1989) 1. DETERMINATION OF SUBCELLULAR DISTRIBUTION OF MSA-63 To determine i f the antig e n r e a c t i n g with HS-63 was a s o l u b l e or i n t e g r a l membrane p r o t e i n s , mouse t e s t e s were homogenized, i n an i s o t o n i c PBS containing. 1. mM PMSF i n the absence of detergent. The s o l u b l e p r o t e i n i n the supernatant and the i n t e g r a l membrane p r o t e i n i n the p e l l e t were separated by c e n t r i f u g a t i o n . The m a j o r i t y of HS-63-reactive ant i g e n was d e t e c t e d i n the s o l u b l e f r a c t i o n when analyzed by i n d i r e c t immunofluorescent i n h i b i t i o n assay. F u r t h e r e x t r a c t i o n of the p e l l e t (membrane f r a c t i o n ) with the same b u f f e r r e s u l t e d i n a r e l e a s e of more a c t i v e a n t i g e n . A f t e r s e v e r a l e x t r a c t i o n s with the same b u f f e r , the a n t i g e n i n the membrane f r a c t i o n c o u l d be completely removed. However, the immunoactivity o f the a n t i g e n i n the s o l u b l e f r a c t i o n was not removed by p a s s i n g through a 0.22 uM M i l l i p o r e f i l t e r membrane. 84 2. PURIFICATION OF MSA-6 3 ANTIGEN Since the immunoactivity of a n t i g e n r e a c t i v e t o HS-63 was shown t o be i n the s o l u b l e f r a c t i o n of mouse t e s t e s homogenate, ammonium s u l f a t e f r a c t i o n a t i o n f o l l o w e d by DEAE i o n exchange chromatography were used t o p u r i f y the corres p o n d i n g a n t i g e n (MSA-63) from mouse t e s t e s . The immunoactivity of a n t i g e n i n each p u r i f i c a t i o n s t e p was measured by i n d i r e c t immunofluorescent i n h i b i t i o n assay. During ammonium s u l f a t e f r a c t i o n a t i o n , the immunoactivity of arttigen was p r e c i p i t a t e d i n the presence of 400 mg/ml ammonium s u l f a t e . A f t e r d i a l y s i s , the f r a c t i o n a t e d MSA-63 was then a p p l i e d t o a D E A E - c e l l u l o s e column. The e l u t i o n p r o f i l e s of p r o t e i n and immunoactivity of t h i s a n t i g e n i n a s a l t g r a d i e n t (0 - 1.0 M NaCl) were shown i n F i g u r e 4. The h i g h e s t immunoactivity was e l u t e d a t 0.38 M of NaCl. Immunoaffinity chromatography was used as the l a s t s t e p f o r the p u r i f i c a t i o n . The b i n d i n g and washing c o n d i t i o n s between the a n t i g e n and immobilized HS-63 antibody were s t u d i e d p r i o r t o the a n t i g e n e l u t i o n . PBS c o n t a i n i n g 0.5 M NaCl was f i n a l l y s e l e c t e d as a washing s o l u t i o n t o remove a l l the n o n s p e c i f i c a l l y bound p r o t e i n s . R e s u l t s f o r the p u r i f i c a t i o n of MSA-6 3 from mouse t e s t i s were summarized i n Table 5. By u s i n g the procedures 85 F i g u r e 4. F r a c t i o n a t i o n of the s o l u b l e supernatant of crude mouse t e s t e s homogenate by D E A E - c e l l u l o s e chromatography. The heavy s o l i d l i n e r e p r e s e n t s NaCl s a l t g r a d i e n t (0-1.0 M). (—) and (—) i n d i c a t e the p r o t e i n c o n c e n t r a t i o n and r e l a t i v e immunoactivity of a n t i g e n , r e s p e c t i v e l y . 86 NaCKM) T a b l e 5. P u r i f i c a t i o n of MSA-63 from Mouse T e s t i s by Two P u r i f i c a t i o n P r o c e d u r e s T o t a l volume P r o t e i n (ml) c o n c e n t r a t i o n (mg/ml) S p e c i f i c a T o t a l Y i e l d a c t i v i t y a c t i v i t y (%) I. S o l u b l e E x t r a c t i o n Supernatant of homogenates 70 Ammonium s u l f a t e p r e c i p i t a t i o n 60 DEAE i o n exchange 200 Immunoaffinity chromatography 1.3 26.6 13.2 0.7 0.23 0.6 1.2 2.3 600 1117 950 392 233 100 85 35 21 I I . D e t e r g e n t E x t r a c t i o n Supernatant 100 of homogenate Immunoaffinity 1 chromatography 100 0.5 0.4 320 4000 160 100 a S p e c i f i c immunoactivity o f MSA-63 was determined by the i n d i r e c t immunofluorescent i n h i b i t i o n assay as d e s c r i b e d i n the s e c t i o n of M a t e r i a l s and Methods ( L i u e t a l . , 1989) d e s c r i b e d , more than a one thousand f o l d p r o t e i n p u r i f i c a t i o n was achieved. On the average, about 0.3 mg of p u r i f i e d a n t i g e n c o u l d be o b t a i n e d from 10 g of mouse t e s t e s . When s t o r e d a t -20°C, p r e c i p i t a t i o n was observed i n the p u r i f i e d MSA-63 an t i g e n p r e p a r a t i o n . In order t o improve the s o l u b i l i t y of MSA-63 an t i g e n , T r i t o n X-100 was used d u r i n g the a n t i g e n p u r i f i c a t i o n . The crude t e s t e s homogenate was f i r s t s o l u b i l i z e d with 2 % T r i t o n X-100. F o l l o w i n g c e n t r i f u g a t i o n , the s o l u b l e supernatant was a p p l i e d d i r e c t l y t o the HS-63 immunoaffinity column. The immunoactivity of the p u r i f i e d MSA-63 was a l s o d e t e c t e d by u s i n g i n d i r e c t immunofluorescent i n h i b i t i o n assay. R e s u l t s of the p u r i f i c a t i o n of MSA-63 from mouse t e s t i s w ith T r i t o n X-100 are summarized i n Table 5. On the average, about an 800 t o 1600 f o l d p r o t e i n p u r i f i c a t i o n was achieved. The p u r i f i c a t i o n data a l s o r e v e a l e d t h a t about 0.5 mg of p u r i f i e d a n t i g e n c o u l d be recovered from 10 g of mouse t e s t e s . The p u r i f i e d MSA-63 r e t a i n e d i t s a b i l i t y t o i n h i b i t HS-63 b i n d i n g t o the methanol-fixed sperm. F o l l o w i n g a t y p i c a l ELISA procedure, the dose-dependent b i n d i n g s between HRP-labeled HS-63 and MSA-63 coated on m i c r o w e l l s were c l e a r l y demonstrated. In 89 c o n t r a s t , no evidence of such b i n d i n g was observed between HRP-l a b e l e d HS-63 and BSA coated on w e l l s . The s p e c i f i c b i n d i n g between HRP-labeled HS-63 and p u r i f i e d MSA-63 coated on mi c r o w e l l s i s shown i n F i g u r e 5. B^ CHARACTERIZATION OF MSA-63 ANTIGENS 1. ANALYSIS OF PURIFIED MSA-63 BY TWO-DIMENSIONAL SDS-GEL (2D-GEL) ELECTROPHORESIS F o l l o w i n g i s o e l e c t r i c f o c u s i n g and SDS g e l e l e c t r o p h o r e s i s , the p u r i f i e d MSA-63 an t i g e n from e i t h e r p u r i f i c a t i o n procedures r e v e a l e d q u i t e a few p r o t e i n spots on the 2D-gels. By u s i n g the T r i t o n X-100 e x t r a c t i o n method, p u r i f i e d MSA-63 d i s p l a y e d a group of p r o t e i n spots w i t h p i s ranging from 4.0 to 6.0 and molecular s i z e s from 20 Kd t o 84 Kd. The 2D-gel of t h i s p r e p a r a t i o n f o r p u r i f i e d MSA-63 i s shown i n F i g u r e 6A. Based on d i s t r i b u t i o n of t h e i r r e s p e c t i v e p i ' s v a l u e and molecular weights, p r o t e i n spots on 2D-gel were s u b d i v i d e d i n t o f i v e groups (see F i g u r e 6A). 90 F i g u r e 5. ELISA showing the s p e c i f i c r e a c t i o n of HRP l a b e l e d HS-63 monoclonal antibody t o the p u r i f i e d MSA-63. Absorbance a t 450 nm was p l o t t e d as a f u n c t i o n of antibody d i l u t i o n . (•) and (0) r e p r e s e n t the absorbance f o l l o w i n g b i n d i n g of HS-63 t o m i c r o t i t e r p l a t e coated with p u r i f i e d MSA-63 and BSA, r e s p e c t i v e l y . D e t a i l e d experimental c o n d i t i o n s are d e s c r i b e d i n the s e c t i o n on M a t e r i a l s and Methods 91 o F i g u r e 6. Two-dimensional g e l e l e c t r o p h o r e s i s o f p u r i f i e d MSA-63 from mouse t e s t e s . The i s o e l e c t r i c p o i n t ( p i ) i s i n d i c a t e d on the top and molecular weight (KD) i s i n d i c a t e d on the r i g h t s i d e of the g e l . (A). P u r i f i e d MSA-63 from T r i t o n X-100 e x t r a c t i o n was s t a i n e d by Coomassie blue R-250. The p r o t e i n spots were s u b d i v i d e d i n t o 5 groups as i n d i c a t e d . (B). P u r i f i e d MSA-63 from s o l u b l e e x t r a c t i o n was s t a i n e d by s i l v e r reagent. (C). S i l v e r s t a i n e d p r o t e i n d i s t r i b u t i o n p a t t e r n of d e t e r g e n t - p u r i f i e d MSA-63 f o l l o w i n g i n c u b a t i o n a t 37°C f o r 5 days with 0.01 % NaN 3. (D). Western b l o t a n a l y s i s of d e t e r g e n t - p u r i f i e d MSA-63 with r a b b i t a n t i s e r a a g a i n s t p r o t e i n spots i n group 3 (MSA-63 p r o t e i n ) as a probe. 93 94 By u s i n g the s o l u b l e e x t r a c t i o n procedure f o r MSA-63 p u r i f i c a t i o n , fewer p r o t e i n spots were d e t e c t e d on the 2D-gel. Only p r o t e i n group 2 and 3 were found on the 2D-gel w i t h p i ' s ran g i n g from 4.0. t o 5.5 and molecular weights r a n g i n g from 24 t o 45 KD ( F i g u r e 6B). The p r o t e i n d i s t r i b u t i o n p a t t e r n s of s o l u b l e - and d e t e r g e n t -p u r i f i e d MSA-63 on 2D-gels were found t o a l t e r s i g n i f i c a n t l y f o l l o w i n g long i n c u b a t i o n at 37°C. A f t e r 5 days i n c u b a t i o n a t 37°C i n the presence of 0.01 % NaN 3, the p r o t e i n spots i n group 1, 2 and 5 were found t o disappear on 2D-gel. The i n t e n s i t y of p r o t e i n spots i n group 4 became very weak when d e t e c t e d by s i l v e r s t a i n i n g reagent. S e v e r a l p r o t e i n spots with s i z e of around 10 to 20 KD appeared on 2D-gel. However, the p r o t e i n spots i n group 3 were not found t o change d u r i n g the i n c u b a t i o n . The 2D-gel a n a l y s i s of t h i s o b s e r v a t i o n i s demonstrated i n F i g u r e 6C. F o l l o w i n g 2 weeks i n c u b a t i o n , a l l p r o t e i n spots on 2D-gel disappeared except those i n group 3 with concomitant i n c r e a s e i n s t a i n i n g i n t e n s i t y o f those of 10 t o 20 KD p r o t e i n s p o t s . The a l t e r a t i o n of g e l p a t t e r n s f o r p u r i f i e d MSA-63 was not s i g n i f i c a n t l y a f f e c t e d by the a d d i t i o n of the f o l l o w i n g protease i n h i b i t o r s i n the i n c u b a t i o n mixture: EDTA (10 mM), iodoacetamide 95 (70 mM), PMSF (1 mM), l e u p e p t i n (1 ug/ml), a n t i p a i n (2 ug/ml), benzamidine (10 ug/ml), chymostatin (1 ug/ml) and p e p s t a i n (1 ug/ml). 2. PRIMARY STRUCTURE ANALYSIS OF PROTEIN SPOTS OF MSA-63 ANTIGEN ON 2D-GELS In view of the h e t e r o g e n e i t y of p u r i f i e d MSA-63 p r o t e i n on 2D-gels, i t i s imperative t o analyze the s t r u c t u r a l and immunological r e l a t i o n s h i p s among these p r o t e i n s p o t s . The p r o t e i n spots of each group were removed from Western b l o t of 2D-g e l s and used f o r amino a c i d sequence a n a l y s i s . F o l l o w i n g t r y p s i n d i g e s t i o n of i n d i v i d u a l p r o t e i n s p o t s , the p e p t i d e fragments were separated by HPLC. The s e p a r a t i o n p r o f i l e s of HPLC are shown on F i g u r e 7. The amino a c i d sequences of the i s o l a t e d p e p t i d e s o b t a i n e d by u s i n g Edman degradation sequencing method are summaried i n Table 6. Through computer a n a l y s i s from the p r o t e i n data bank, the amino a c i d sequences from t r y p s i n - d i g e s t e d p e p t i d e d e r i v e d from p r o t e i n spots i n group l , 2 and 4 showed 100 % sequence homology F i g u r e 7. High-performance l i q u i d chromatography on r e v e r s e phase C-18 column of t r y p s i n - d i g e s t e d p u r i f i e d MSA-63 a n t i g e n . A c e t o n i t r i l e (0-70 %) g r a d i e n t i n 0.1 % t r i f l u o r o a c e t i c a c i d (TFA) was used as the mobile phase. The column was'performed a t a flow r a t e of 2.0 ml/min. The s e l e c t e d - p e p t i d e peaks f o r amino a c i d sequence d e t e r m i n a t i o n were i n d i c a t e d by arrows. (A) HPLC p r o f i l e of t r y p t i c p e p t i d e s from p r o t e i n spots i n group 1 and 2. (B) HPLC p r o f i l e of t r y p t i c p e p t i d e s from p r o t e i n spots i n group 3. (C) HPLC p r o f i l e of t r y p t i c p e p t i d e s from p r o t e i n spots i n group 4. (D) HPLC p r o f i l e of t r y p t i c p e p t i d e s from MSA-63 p r o t e i n p u r i f i e d a c c o r d i n g t o the s o l u b l e e x t r a c t i o n procedure. (Work c a r r i e d out i n Dr. Ruedi Aebersold's l a b i n Biomedical Research Center a t UBC) 97 A i Time (0---120min) Time (0---120min) 98 99 T a b l e 6. Amino a c i d sequence a n a l y s i s of p u r i f i e d MSA-63. P r o t e i n s p o t Peak Amino a c i d sequence 9 Sequence (Group) No. homology ti 1, 2 14 X-Tyr-Ser-Phe-Thr-Thr-Thr-Ala-Glu oC-actin 3 4 Tyr-Glu-Thr-Phe-Leu-Asp-Glu N i l G ly-Glu-Gly-Val-X-Thr-Thr-Gln-Asn-Ser-Gln N i l Met-Gln-Ile-Met N i l 4 10 I l e - I l e - A l a - P r o - P r o - G l u c t - a c t i n Leu-Asp-Leu-Ala-Gly-Arg o t-actin 12 Tyr-Ser-Phe-Thr-Thr-Thr-Ala-Glu-Arg o i - a c t i n X-X-Glu-Ala-Phe-Leu-Asn-Phe-Lys o*.- a c t i n 20 Leu-Thr-Asp-Tyr-Met o i - a c t i n 27 Tyr-Ala-Asn-Thr-Val-Leu-Ser-Gly-Gly- o(.-actin -Thr-Thr-Met-Tyr-Phe-Gly-Ile-Ala-Asp S o l u b l e -p u r i f i e d 18 Phe-Met-Val-Gln-Gly-X-Glu-Asn-Met-Phe- N i l MSA-63 -Pro-Ser-Met-Asn-Leu-Phe a X : u n i d e n t i f i e d amino a c i d r e s i d u e . t o those of the o ( - a c t i n p r o t e i n . Three amino a c i d sequences of pep t i d e s obtained from t r y p s i n - d i g e s t e d p r o t e i n spots i n group 3 were shown t o be u n r e l a t e d to any oth e r p u b l i s h e d p r o t e i n s o r pe p t i d e s (Table 6 ) . P u r i f i e d MSA-63 obt a i n e d from the s o l u b l e e x t r a c t i o n procedure was a l s o employed f o r amino a c i d sequence a n a l y s i s of the p e p t i d e s . A f t e r s e p a r a t i o n by u s i n g HPLC, peak 18 pep t i d e s from the t r y p s i n - d i g e s t e d p u r i f i e d MSA-63 was s e l e c t e d f o r the amino a c i d sequence a n a l y s i s ( F i g u r e 7D). The amino a c i d sequence of t h i s p e p t i d e was shown t o have no homology with those of the p u b l i s h e d known p r o t e i n s and p e p t i d e s . Immunological r e l a t i o n s h i p of MSA-63 p r o t e i n spots on 2D-gels was a l s o i n v e s t i g a t e d . The p r o t e i n spots i n group 1, 2 and 3 were a l s o removed s e p a r a t e l y from the Western b l o t of 2D-gel and served as immunogen f o r immunizing r a b b i t s . The r a i s e d a n t i s e r a ( a f t e r 5 immunizations) were then used as probes f o r f u r t h e r Western b l o t a n a l y s i s . A n t i s e r a r a i s e d a g a i n s t p r o t e i n spots, i n groups 1 and 2 c o u l d mutually c r o s s - r e a c t with each ot h e r , but f a i l e d t o re c o g n i z e p r o t e i n spots i n group 3. However, the a n t i s e r a a g a i n s t p r o t e i n spots i n group 3 c o u l d c r o s s - r e a c t w i t h 101 a l l p r o t e i n spots on the 2D-gel ( F i g u r e 6D). Furthermore, the a n t i s e r a a g a i n s t p r o t e i n spots i n group 1 c o u l d h i g h l y c r o s s - r e a c t w i t h o C-actin from r a b b i t muscle (Sigma A2522) by u s i n g dot b l o t assay. However, r a b b i t a n t i s e r a a g a i n s t p r o t e i n spots i n group 2 and 3 r e v e a l e d no c r o s s - r e a c t i v i t y t o o(-a c t i n . By u s i n g an radioimmunobinding method, HS-63 monoclonal antibody was shown t o have no s i g n i f i c a n t c r o s s - r e a c t i v i t y t o cL-a c t i n from r a b b i t muscle. By means of i n d i r e c t immunofluorescent assay, o n l y a n t i s e r a r a i s e d a g a i n s t p r o t e i n spots i n group 3 were shown t o r e a c t w i t h the acrosome of mouse sperm f i x e d on s l i d e s . The sperm s t a i n i n g p a t t e r n was s i m i l a r t o t h a t of HS-63. 3. MOLECULAR WEIGHT ANALYSIS OF PURIFIED MSA-63 PROTEINS To analyze the molecular s i z e d i s t r i b u t i o n of MSA-63 antig e n i n n a t i v e form, the p u r i f i e d MSA-63 an t i g e n o b t a i n e d from T r i t o n X-100 e x t r a c t i o n method was a p p l i e d t o a Sephacryl S-300 g e l f i l t r a t i o n column. In the presence of 0.1 % T r i t o n X-100, a broad d i s t r i b u t i o n of MSA-6 3 immunoactivity was observed with a maximum 102 a c t i v i t y i n the molecular weight r a n g i n g from 200 Kd t o 300 Kd (Fi g u r e 8 ) . Antigen f r a c t i o n s w i t h the h i g h e s t immunoactivity were subsequently analyzed by SDS-PAGE. I t was found t h a t the mole c u l a r weight of major p r o t e i n bands on the g e l ranged between 24 Kd and 50 Kd ( F i g u r e 8 ) . 4. INTERACTION BETWEEN MSA-63 AND ACTIN In view of hig h molecular weight d i s t r i b u t i o n o f MSA-63 immunoactivity i n n a t i v e form, the i n t e r a c t i o n between MSA-63 and u i - a c t i n (from r a b b i t muscle, Sigma A2522) was i n v e s t i g a t e d . F o l l o w i n g o v e r n i g h t i n c u b a t i o n w i t h p u r i f i e d MSA-63 (prepared by T r i t o n e x t r a c t i o n method) a t 4°C, the molecular weight d i s t r i b u t i o n p r o f i l e of a c t i n was found t o change when analyzed by Sephacryl S-300 g e l f i l t r a t i o n ( F i g u r e 9 ) . 5. ANALYSIS OF PURIFIED MSA-63 PROTEINS BY ENZYMATIC DEGLYCOSYLATION Attempts were made t o analyze the molecular h e t e r o g e n e i t y of MSA-6 3 p r o t e i n though enzymatic d e g l y c o s y l a t i o n . N - g l y c o s i d a s e and O-glycosidase were used f o r i n v i t r o d e g l y c o s y l a t i o n of MSA-63 p r o t e i n s . A f t e r o v e r n i g h t i n c u b a t i o n a t 37°C, the p r o t e i n 103 F i g u r e 8. Sephacryl S-300 g e l f i l t r a t i o n chromatography and SDS-PAGE t o r e v e a l the molecular s i z e d i s t r i b u t i o n of p u r i f i e d MSA-63 an t i g e n . Arrows A, B, and C i n d i c a t e the p o s i t i o n of molecular s i z e marker of 670, 158, and 44 KD, r e s p e c t i v e l y . The p r o f i l e of an t i g e n immunoactivity determined by i n d i r e c t immunofluorescent i n h i b i t i o n assay i n d i c a t e d the main a c t i v i t y was det e c t e d i n f r a c t i o n s with molecular s i z e r anging from 200-300 Kd. The corresp o n d i n g f r a c t i o n s examined by SDS-PAGE are shown on the top. The molecular marker i s i n d i c a t e d on the r i g h t s i d e of the g e l . 104 Fraction NO. 13 15 17 19 21 23 25 27 29 10 15 20 Fraction NO. - 4 7 K d - 3 3 Kd -24Kd 25 F i g u r e 9. Sephacryl S-300 g e l f i l t r a t i o n chromatography t o r e v e a l the i n t e r a c t i o n between - a c t i n and p u r i f i e d MSA-63. (-o-) i n d i c a t e s the molecular weight d i s t r i b u t i o n of 1 2 5 I - l a b e l e d A c t i n p r o t e i n ( s p e c i f i c a c t i v i t y 0.02 mCi/ug). (-X-) i n d i c a t e s the molecular weight d i s t r i b u t i o n of 1 2 5 I - l a b e l e d a c t i n p r o t e i n incubated with 10 ug of p u r i f i e d MSA-63 f o r o v e r n i g h t a t 4°C. The f r a c t i o n a t e d r a d i o a c t i v i t y i s measured by u s i n g a -counter. Arrows A, B, and C i n d i c a t e the p o s i t i o n of estimated molecular s i z e m a r k e r of 670, 158, and 44 KD, r e s p e c t i v e l y . 106 Fraction No. d i s t r i b u t i o n p a t t e r n o f MSA-6 3 on SDS-gel was not changed i n the presence of e i t h e r N - g l y c o s i d a s e or O - g l y c o s i d a s e . R e s u l t s of t h i s a n a l y s i s are presented i n F i g u r e 10. Human c h o r i o n i c gonadotrophin (hCG) p r o t e i n served as p o s i t i v e c o n t r o l t o show the enzymatic a c t i v i t y o f these two g l y c o s i d a s e . PRODUCTION AND EVALUATION OF ANTISERA ON FERTILIZATION Female mice and r a b b i t s were immunized wi t h p u r i f i e d MSA-63 ant i g e n t o i n v e s t i g a t e immunogenicity and a n t i f e r t i l i t y e f f e c t s . MSA-6 3 ant i g e n prepared by the s o l u b l e e x t r a c t i o n procedure was used as a immunogen t o r a i s e a n t i s e r a i n both mice and r a b b i t s . The t i t r e of a n t i s e r a i n c r e a s e d with i n c r e a s i n g number of immunizations. A n t i 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 MSA-63 from r a b b i t and mouse were shown to r e a c t with the acrosome of mouse and human sperm when examined by i n d i r e c t immunofluorescent assay. The s p e c i f i c i t y of these a n t i s e r a a g a i n s t MSA-63 was a l s o demonstrated by u s i n g ELISA and compared with t h a t of HS-63 and an u n r e l a t e d antibody. R e s u l t s of t h i s study are presented i n F i g u r e 11. 108 F i g u r e 10. (A). Enzymatic d e g l y c o s y l a t i o n o f MSA-63 p r o t e i n s u s i n g N - g l y c o s i d a s e and O-glycosidase i n the presence of 1% T r i t o n X-100 and 0.1 % SDS i n 0.1 phosphate b u f f e r pH 7.3. 1 2 5 i -l a b e l e d MSA-63 p r o t e i n ( s p e c i f i c a c t i v i t y 0.1 mCi/ug) was incubated w i t h (1) 0.6 u n i t of N- g l y c o s i d a s e (2) 1 mU of O-g l y c o s i d a s e (3) 0.6 u n i t of N-gly c o s i d a s e p l u s 1 mU of O T g l y c o s i d a s e (4) no enzyme f o r o v e r n i g h t a t 37°C. The p r o t e i n p a t t e r n of each treatment was analyzed by SDS-PAGE. Molecular Weight (KD) i s i n d i c a t e d on the r i g h t s i d e of the g e l . (B). Enzymatic d e g l y c o s y l a t i o n of hCG p r o t e i n was t r e a t e d under the same c o n d i t i o n s e r v i n g as p o s i t i v e c o n t r o l . 109 110 F i g u r e 11. ELISA showing the b i n d i n g between HS-63, mouse isoimmunesera, r a b b i t anti-MSA-63 s e r a or u n r e l a t e d a n t i b o d i e s and the p u r i f i e d MSA-63 coated on m i c r o w e l l s . Absorbance a t 450 nm was p l o t t e d as a f u n c t i o n of antibody d i l u t i o n i n l o g s c a l e , where ( k ) denotes HS-63, (O) i s mouse isoimmune s e r a a g a i n s t MSA-6 3 , ( •) denotes r a b b i t a n t i s e r a a g a i n s t MSA-63 and (X) i s u n r e l a t e d anti-hCG monoclonal antibody. I l l tuu 09V 0 0 112 The a n t i f e r t i l i t y e f f e c t of these a n t i s e r a was e v a l u a t e d by u s i n g a mouse i n v i t r o f e r t i l i z a t i o n experiment (Table 7 ) . A s i g n i f i c a n t l y lower f e r t i l i z a t i o n r a t e was observed i n the presence of mouse isoimmune s e r a or r a b b i t anti-MSA-63 as compared t o t h a t of the c o n t r o l u s i n g nonimmune s e r a . The a n t i f e r t i l i t y e f f e c t of r a b b i t a n t i s e r a r a i s e d a g a i n s t MSA-63 was a l s o e v a l u a t e d by i n v i v o mating experiments f o l l o w i n g p a s s i v e immunizations. Under the same experimental c o n d i t i o n s , p o l y c l o n a l a n t i s e r a appeared t o be more e f f e c t i v e than HS-63 monoclonal antibody i n terms, of i n v i v o f e r t i l i z a t i o n i n h i b i t i o n . R e s u l t s of such a n a l y s i s are presented i n Table 4. 113 T a b l e 7. I n h i b i t o r y e f f e c t of p o l y c l o n a l a n t i s e r a r a i s e d a g a i n s t MSA-63 on i n v i t r o f e r t i l i z a t i o n of mouse oocytes. A n t i s e r a a Number of Ova examined Ova f e r t i l i z e d F e r t i l i z a t i o n P v a l u e assays (No.) (No.) r a t e (%) C o n t r o l (NMS) 70 57 81.4 MSA-63 C o n t r o l (NRS) 3 2 54 55 16 28 29.6 50.9 < o . o o r RSA-63 61 15 24 .6 <0.01 c a A n t i s e r a used f o r experiments: NMS: nonimmune mouse serum, MSA-63: isoimmune s e r a a g a i n s t MSA-63, NRS: nonimmune r a b b i t serun, RSA-63: r a b b i t anti-MSA-63. ^Compared wi t h the c o n t r o l group (NMS). cCompared wi t h the c o n t r o l group (NRS). P a r t I I I . CLONING OF MSA-63 CDNA AND PRODUCTION OF ITS FUSION PROTEIN A^ IMMUNOSCREENING OF MOUSE TESTIS cDNA LIBRARY Rabbit a n t i s e r a r a i s e d a g a i n s t the MSA-63 p r o t e i n s were used f o r the immunoscreening of a mouse t e s t i s cDNA l i b r a r y t o d e t e c t the cDNA c l o n e s which expressed the corresponding f u s i o n p r o t e i n s . A mouse t e s t i s cDNA l i b r a r y c o n t a i n i n g approximately 1 x 10 6 independent recombinant bacteriophages was c o n s t r u c t e d i n a lambda g t l l e x p r e s s i o n v e c t o r . Through the immunoscreening of 6 x H • • • • 10 bacteriophages from t h i s l i b r a r y , more than 60 p o s i t i v e primary c l o n e s were i d e n t i f i e d ( F i g u r e 12). Twelve of these p o s i t i v e primary c l o n e s were s e l e c t e d randomly.for secondary immunoscreening. The DNA of these p u r i f i e d recombinant bacteriophages was analyzed by u s i n g Southern b l o t h y b r i d i z a t i o n . Two of the cDNA i n s e r t s were randomly s e l e c t e d as probes. Among the p o s i t i v e primary c l o n e s , s i x c o u l d c r o s s - h y b r i d i z e with each o t h e r . Under h i g h l y s t r i n g e n t washing c o n d i t i o n s , f o u r of the c r o s s - h y b r i d i z e d c l o n e s were i d e n t i f i e d ( F i g u r e 13). 115 F i g u r e 12. Immunoscreening of mouse t e s t i s cDNA l i b r a r y by u s i n g r a b b i t anti-MSA-63 s e r a as the d e t e c t i n g probe. P o s i t i v e c l o n e s are c i r c l e d . (Work c a r r i e d out i n Dr. C h r i s Lau's l a b a t U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o ) 116 117 F i g u r e 13. Southern b l o t a n a l y s i s t o demonstrate the c r o s s h y b r i d i z a t i o n of immuno-screened p o s i t i v e c l o n e s . (A). cDNA i n s e r t s o f Clone 3, 10 and 12 are shown to h y b r i d i z e with the cDNA probe from c l o n e 6. (B). Using the cDNA i n s e r t of c l o n e 12 as a probe, cDNA of c l o n e 3 ,6 and 10 are shown t o h y b r i d i z e with the probe. Hind I I I d i g e s t e d lambda DNA as s i z e marker shown on the l e f t , are 23.13 Kb, 9.41 Kb, 6.55 Kb, 4.36 Kb, 2.32 Kb, 2.02 Kb and 0.56 Kb, r e s p e c t i v e l y from the top. (Work c a r r i e d out i n Dr. C h r i s Lau's l a b a t U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o ) (The data were p u b l i s h e d i n L i u e t a l . , 1990) 118 23.13 9.41 6.55 4.36 2.32 2.02 0.56 z * '§ 11 10 12 B — M M — 23.13 _ 9.41 _ 6.55 ~ 4.36 -2.32 2.02 0.56 "* 10 12 119 When analyzed by agarose g e l e l e c t r o p h o r e s i s , the s i z e of the cDNA i n s e r t of clone 6 and 12 were determined t o be 0.8 and 1.0 Kb, r e s p e c t i v e l y . These two cDNA cl o n e s were s e l e c t e d f o r pr o d u c t i o n of recombinant f u s i o n p r o t e i n s . B. SCREENING OF THE MOUSE TESTIS cDNA LIBRARY BY USING THE CLONED  DNA PROBE The cDNA i n s e r t of cl o n e 12 was used as a probe t o screen the mouse t e s t i s cDNA l i b r a r y i n order t o i s o l a t e l a r g e r or f u l l l e n g t h c l o n e s . A f t e r s c r e e n i n g of 1 X 10 5 bacteriophages, s e v e r a l p o s i t i v e c l o n e s were i d e n t i f i e d . By Southern b l o t a n a l y s i s , the molecular s i z e of many c l o n e s was about 1.2 Kb ( F i g u r e 14A). The r e s t r i c t i o n maps of these cDNA cl o n e s were found t o be i d e n t i c a l ( F i g u r e 14B). One of these c l o n e s w i t h a s i z e of about 1.2 Kb was used f o r DNA sequence a n a l y s i s . 120 F i g u r e 14. (A). Southern b l o t assay of e s t a b l i s h e d p o s i t i v e cDNA clo n e s by u s i n g c l o n e 12 cDNA as a probe. (B). The r e s t r i c t i o n map a n a l y s i s of e s t a b l i s h e d p o s i t i v e cDNA c l o n e s d i g e s t e d with EcoRI and P s t l . The molecular s i z e markers i n Kb are i n d i c a t e d on the l e f t . 121 122 DNA SEQUENCE ANALYSIS F o l l o w i n g EcoR I d i g e s t i o n , the cDNA i n s e r t was o b t a i n e d from the recombinant phages and then subcloned i n t o plasmid pUC18. The r e s t r i c t i o n map of the cDNA i n s e r t was deduced from the a n a l y s i s of r e s t r i c t i o n enzyme d i g e s t i o n . Pst I d i g e s t e d fragments were c o n t i n u o u s l y subcloned i n t o pUC18 and s u b j e c t e d to DNA sequencing a n a l y s i s . The r e s t r i c t i o n map and sequencing s t r a t e g y f o r the cDNA i n s e r t of MSA-63 gene are presented i n F i g u r e 15. The n u c l e o t i d e sequence of the cDNA i n s e r t and the c o r r e s p o n d i n g p r o t e i n sequence are presented i n Figure. 16. The cDNA i n s e r t was 1067 bp i n l e n g t h and c o n t a i n e d an open r e a d i n g frame of 783 n u c l e o t i d e s (261 amino a c i d s ) s t a r t i n g from an i n i t i a t i o n codon (ATG) at n u c l e o t i d e s 60-62. The t e r m i n a t i o n codon TGA was l o c a t e d a t n u c l e o t i d e s 842-844. The Length of the 5' u n t r a n s l a t e d r e g i o n was found t o be 59 bp and n u c l e o t i d e s upstream from the ATG i n i t i a t i o n codon were AAATCAAA, being s i m i l a r t o most e u k a r y o t i c s t a r t codons f o r t r a n s l a t i o n (Kozak, 1984) . 123 F i g u r e 15. R e s t r i c t i o n map and sequencing s t r a t e g y f o r the cDNA i n s e r t of MSA-63. R e s t r i c t i o n s i t e s are shown at the t o p : H: Hind I I I , P: P s t I, B: B'gl I I , X: Xbal I. EcoR I and P s t I are used f o r s u b c l o n i n g i n t o pUC18. Arrows i n d i c a t e the d i r e c t i o n and extent o f each sequence d e t e r m i n a t i o n . 124 ID CN o 125 F i g u r e 16. The n u c l e o t i d e sequence and deduced amino a c i d sequence of MSA-63. The p r e d i c t e d amino a c i d sequence i s shown below the n u c l e o t i d e sequence. The numbering t o the r i g h t i n d i c a t e s the n u c l e o t i d e and amino a c i d p o s i t i o n s . A p o l y A a d d i t i o n s i g n a l i s underscored with the symbol (***) and an mRNA consensus degradation sequence i s underscored with the symbol ( A A A ) . The p r e d i c t e d a n t i g e n i c determinants are underscored with the symbol (...) and a c a n o n i c a l N - l i n k e d g l y c o s y l a t i o n sequences i s underscored with the symbol (+). The u n d e r l i n e d sequence i s i d e n t i c a l t o t h a t from amino a c i d sequencing a n a l y s i s of p e p t i d e s i s o l a t e d from t r y p s i n - d i g e s t e d MSA-63 p r o t e i n . 126 5' CAGTTTCTTC TCAGCTCTTG AGTGTGCCAC ATTAGAGATC TTTATTTACC TAAATCAAA ATG AAG GAG TTA ATC TTA CTG GGA 83 m m Met Lys Glu Leu Ilu Leu Leu Gly 8 GGT CTT TAT CTG CTT TCT GCC CAA GGA GCA CCA CCA GGT CAG CCT GAG GAG CTT CTT GAC TCT GTA GAC CAA 155 Gly Leu Tyr Leu Leu Ser Ala Gin Gly Ala Pro Pro Gly Gin Pro Glu Glu Leu Leu Asp Ser Val Asp Gin 32 CAA GCT TCG GTT CAG CAA CTT TCA AGC GAG TAT CTC TCA CTC GCA AAC OT TCA GAT GCC GAG GCT TTA TAT 227 Gin Ala Ser Val Gin Gin Leu Ser Ser Glu Tyr Leu Ser Leu Ala Asn Pro Ser Asp Ala Glu Ala Leu Tyr 56 GAA ACT CCT TTA GAT GAG AAG ACT CTG AGI GGT CAT AGT TCA AGI GAA CAG GAA TCA AGT GAG CAT GCT GTA 299 Glu Thr Pro Leu Asp Glu Lys Thr Leu Ser Gly His Ser Ser Ser Glu Gin Glu Ser Ser Glu His Ala Val 80 G f l G A A ( ^ T f l«a ( C T ( & C A C r c T T C A G U G A A 371 Ala Glu His Ser Ala Gly Glu His Ser Ser Gly Glu Gin Ser Ser Glu His Net Ser Gly Asp His Met Ser 104 QGAGAGMTTGTCAGMCACACTTCAGAGGAGCACITCTO 443 Gly Glu His Leu Ser Glu His Thr Ser Glu Glu His Ser Ser Gly Glu His Thr Ser Thr Glu His Thr Ser 128 GGT GAA CAA CCT GCA A C T G A A ( ^ T f £ T C A A G T G A C C A G C C C T C C G A A G Q T C T T C ! A 515 Gly Glu Gin Pro Ala Thr Glu Gin Ser Ser Ser Asp Gin Pro Ser Glu Ala Ser Ser Gly Glu Val Ser Gly 152 GACGAAGCAGGTGAACAGGIGTCTAGCMACAAATGACAAAGAAAATGATG(5ATGAGTAtt 587 Asp Glu Ala Gly Glu Gin Val Ser Ser Glu Thr Asn Asp Lys Glu Asn Asp Ala Met Ser Thr Pro Leu Pro 176 AGC ACA TCT GCA GCC ATA ACA TTA AAT TGC CAC ACA TGT GCT TAT ATG AAT GAT GAT GCA AAA TGT CTC CGT 659 Ser Thr Ser Ala Ala He Thr Leu Asn Cys His Thr Cys Ala Tyr Met Asn Asp Asp Ala Lys Cys Leu Arg 200 GGA GAA GGA GTA TGC ACC ACT CAA AAC TCC CAG CAG TGC ATG TTA AAG AAG ATC TTT GAA GGT GGA AAA CTC 731 Gly Glu Gly Val Cys Thr Thr Gin Asn Ser Gin Gin Cys Met Leu Lys Lys He Phe Glu Gly Gly Lys Leu 224 CAG TTC ATG GTT CAA GGG TGT GAG AAC ATG TGC CCA TCT ATG ACC CTC TTC TCT CAT GGA ACA AGA ATG CAA 803 Gin Phe Met Val Gin Gly Cys Glu Asn Met Cys Pro Ser Met Asn Leu Phe Ser His Gly Thr Arg Met Gin 248 ATT ATG TGC TGT CGG AAT GAA CCT CTC TGC AAC AAG GTC TAG ATGCCCGTGC CCTACTTCTT GCTCTGACTT AGGCAGGTTC 885 He Met Cys Cys Arg Asn Glu Pro Leu Cys Asn Lys Val TO! 261 ACCACTCTAC TTGGCTCAAT TTATGTTCAA CTTCAACAAC TAATCACATC GGCTCTGCCT GATCACCAGA TAAGAAGCTC AAACCTTGTC 975 AA AAA 3' TTTATTGATA CCCCATTGCC TATGTCCKT GCCTTACTTT GCTCCCATCC TTCCGCAGAT GTTOTTTTT GCAATAAATT GCTATTAAAGAA 1067 urn 127 Through t h i s sequence a n a l y s i s , a p u t a t i v e e u k a r y o t i c mRNA degradation sequence (ATTTA) was a l s o i d e n t i f i e d a t n u c l e o t i d e s 903-907 (Caput e t a l . , 1986; Shaw and Kamem, 1986) A consensus p o l y a d e n y l a t i o n sequence (AATAAA) a t n u c l e o t i d e s 1048-1053 was a l s o l o c a t e d a t the 3' u n t r a n s l a t e d r e g i o n f o l l o w e d by a p o l y A t a i l . The sequence a n a l y s i s r e v e a l e d t h a t t h e r e was a c o n s i d e r a b l e o v e r l a p between cDNA c l o n e 6 and 12. cDNA cl o n e s 6 and 12 were found t o s t a r t a t n u c l e o t i d e s 68 and 418, r e s p e c t i v e l y . The amino a c i d sequence f o r MSA-6 3 deduced from the cDNA sequence p r e d i c t e d a p r o t e i n of 27.9 Kd. P a r t o f the amino a c i d sequence was found t o be matched with those o b t a i n e d from the s o l u b l e - p u r i f i e d MSA-63 a n t i g e n and from p r o t e i n spots i n group 3 on 2D-gel. The amino a c i d composition of the deduced MSA-63 p r o t e i n i s presented i n Table 8. I t i s noted t h a t MSA-63 c o n s i s t s a r e l a t i v e l y h i g h percentage of s e r i n e (15.7 % ) , glutamic a c i d s (12.2 % ) , and l e u c i n e (8.4 % ) . There i s no tryptophane r e s i d u e i n the e n t i r e sequence. The r e p e a t i n g amino a c i d m o t i f s : Ser-Ser-128 T a b l e 8. Amino a c i d c o m p o s i t i o n of the deduced MSA-63 p r o t e i n . Amino a c i d r e s i d u e Number of r e s i d u e Percentage ( % ) A l a 17 6.5 Arg 3 1.1 Asn 10 3 . 8 Asp 11 4.2 Cys 10 3.8 G i n 18 6.8 G l u 32 12.2 G l y 20 7.6 H i s 13 4 . 9 l i e 4 1.5 Leu 22 8.4 Lys 8 3.0 Met 11 4 . 2 Phe 3 1.1 Pro 11 4.2 Se r 41 15.7 T h r 15 5.7 T r p 0 .0 T y r 4 1.5 V a l 8 3.0 129 G l y - G l u and Ser-Ser-Glu were found i n h i g h frequency i n the e n t i r e amino a c i d r e s i d u e s . A c a n o n i c a l N - l i n k e d g l y c o s y l a t i o n sequence (Asn-Xaa-Ser/Thr) ( P l e s s and Lennarz, 1977; Hart e t a l . , 1979) was l o c a t e d a t amino a c i d r e s i d u e 49 (see F i g u r e 16). Acc o r d i n g t o the PC/GENE computer program a n a l y s i s , the h y d r o p h i l i c i t y p l o t of deduced MSA-63 p r o t e i n showed a hydrophobic amino terminus which i s a c h a r a c t e r i s t i c of a s i g n a l p e p t i d e (von He i j n e , 1986). The main hydrophobic domains were found t o be proximal t o the carboxy terminus. The r e s u l t of t h i s a n a l y s i s i s presented i n F i g u r e 17. Three a n t i g e n i c determinants having the h i g h e s t p o i n t s of h y d r o p h i l i c i t y were:. (1) amino a c i d r e s i d u e s 164-169; Asn-Asp-Lys-Glu-Asn-Asp (2) amino a c i d r e s i d u e s 162-167; Glu-Thr-Asn-Asp-Lys-Glu (3) amino a c i d r e s i d u e s 72-77; Glu-Gln-Glu-Ser-Ser-Glu (see F i g u r e 16). The t h e o r e t i c a l i s o e l e c t r i c p o i n t ( p i ) of MSA-63 p r o t e i n was estimated t o be 4.2. T h i s i s c o n s i s t e n t w i t h the p i value of p r o t e i n spots i n group 3 on 2D-gel. 130 F i g u r e 17. H y d r o p h i l i c i t y p l o t f o r the deduced MSA-63 amino a c i d sequence. Three of the h i g h e s t h y d r o p h i l i c r e g i o n s assumed t o be the a n t i g e n i c determinants of t h i s p r o t e i n , are i n d i c a t e d by arrows on the p l o t . 131 Number of amino a c i d s The search of PC/GENE data base ( r e l e a s e 6.25, J u l y 10, 1990) r e v e a l e d l i t t l e homology of n u c l e o t i d e and amino a c i d sequence t o any known genes or p r o t e i n s . However, about 60 t o 70 % homology i n n u c l e i c a c i d and amino a c i d sequence was observed between MSA-63 and SP-10 a n t i g e n of human sperm o r i g i n (Wright e t a l . , 1990). D. PRODUCTION AND CHARACTERIZATION OF MSA-63 FUSION PROTEIN 1. PREPARATION OF RECOMBINANT MSA-63 FUSION PROTEIN FROM LYSOGENIC STRAIN OF E^. COLI Two of the recombinant phages c a r r y i n g cDNA i n s e r t from c l o n e s 6 and 12, r e s p e c t i v e l y were chosen f o r p r oduction of MSA-63 f u s i o n p r o t e i n i n a l y s o g e n i c s t r a i n of E_s_ c o l i . A f t e r i n f e c t i o n and s e l e c t i o n , s e v e r a l l y s o g e n i c b a c t e r i a c o l o n i e s were formed. MSA-63 f u s i o n p r o t e i n expressed by the recombinant lysogens was d e t e c t e d and c h a r a c t e r i z e d by u s i n g Western b l o t and immunobinding i n h i b i t i o n assay. The SDS g e l s and the corresponding Western b l o t assay of l y s a t e s of Ej. c o l i w ith or without recombinant MSA-6 3 cDNA i n s e r t are shown i n F i g u r e 18. By Western b l o t a n a l y s i s , the 133 F i g u r e 18. SDS-PAGE and Western b l o t assay of recombinant MSA-63 f u s i o n p r o t e i n . P r o t e i n s on SDS-gel s t a i n e d by Coomassie bl u e are shown on lanes 1 and 3. The r e s u l t s of Western b l o t are shown on lanes 2 and 4: Lanes 1 and 2 denote the l y s a t e s of c l o n e 12 lysogens, r e s p e c t i v e l y . A recombinant f u s i o n p r o t e i n d e t e c t e d by r a b b i t anti-MSA-63 s e r a i s shown on lane 2 with the arrow i n d i c a t i n g a molecular s i z e of about 150 KD. Lanes 3 and 4 r e p r e s e n t those of the c o n t r o l and l y s a t e s ' of E . c o l i s t r a i n Y1089, r e s p e c t i v e l y . (The data were p u b l i s h e d i n L i u e t a l . , 1990) 134 1 2 3 4 135 expressed MSA-6 3 f u s i o n p r o t e i n from l y s o g e n i c E^. c o l i c a r r y i n g clone 12 cDNA i n s e r t was d e t e c t e d by u s i n g p o l y c l o n a l a n t i s e r a r a i s e d a g a i n s t n a t i v e MSA-63 p r o t e i n as the probe. The molecular weight of the expressed f u s i o n p r o t e i n was estimated t o be about 150 KD on SDS g e l . F i g u r e 19 shows the im m u n o s p e c i f i c i t y of the MSA-6 3 recombinant f u s i o n p r o t e i n t o HS-63 as demonstrated by radioimmunosorbent i n h i b i t i o n assay. B a c t e r i a l c e l l l y s a t e s c o n t a i n i n g the f u s i o n p r o t e i n (from c l o n e 12 and clo n e 6 lysogens) were found t o i n h i b i t the b i n d i n g of 1 2 5 I - l a b e l e d HS-63 to the mouse sperm coated on mi c r o w e l l s i n a dose-dependent manner. 136 F i g u r e 19. Percent i n h i b i t i o n of the b i n d i n g of -"-"I-labeled HS-63 ( s p e c i f i c a c t i v i t y 0.1 mCi/ug) to mi c r o w e l l s coated w i t h the s o l u b l e e x t r a c t of mouse sperm by the l y s a t e s of cl o n e 6 lysogens (•), c l o n e 12 lysogen (O) and Y1089 host (X). l x l O 6 cpm/well i s used f o r each assay. The i n i t i a l p r o t e i n c o n c e n t r a t i o n of c e l l l y s a t e s was a d j u s t e d t o 5 mg/ml. (The data were p u b l i s h e d i n L i u e t a l . , 1990) 137 0 I L_L_1 I I i U 0 1 2 4 8 16 Dilution of Cell Lysate 138 2 . ISOLATION OF FUSION PROTEINS AND EVALUATION OF CORRESPONDING ANTISERA MSA-63 f u s i o n p r o t e i n from the recombinant lysogens was p a r t i a l l y p u r i f i e d by g e l f i l t r a t i o n chromatography (Sephacryl S-300). F r a c t i o n s c o n t a i n i n g s p e c i f i c immunoactivity t o HS-63 were c o l l e c t e d and used as immunogens t o immunize female mice and r a b b i t s . F o l l o w i n g the t y p i c a l immunization p r o t o c o l s , the mouse and r a b b i t a n t i s e r a were r a i s e d a g a i n s t p a r t i a l l y p u r i f i e d recombinant MSA-63 f u s i o n p r o t e i n . A n t i s e r a r a i s e d a g a i n s t the host b a c t e r i a l e x t r a c t served as the negative c o n t r o l . By u s i n g i n d i r e c t immunofluorescent assay, the a n t i s e r a from mouse and r a b b i t were shown t o r e a c t only with the acrosome of mouse sperm. T h i s r e s u l t i s shown F i g u r e 20. The t i t r e s of mouse a n t i s e r a were i n c r e a s e d t o 1:500 a f t e r the f o u r t h immunization. S p e c i f i c b i n d i n g between the a n t i s e r a and mouse sperm coated on mic r o w e l l s was a l s o demonstrated by ELISA. A n t i s e r a a g a i n s t recombinant M S A-6 3 f u s i o n p r o t e i n s showed s i g n i f i c a n t dose-dependent b i n d i n g t o coated mouse sperm. In c o n t r a s t , a n t i s e r a a g a i n s t host b a c t e r i a l e x t r a c t r e v e a l e d o n l y n o n s p e c i f i c b i n d i n g t o coated w e l l s . R e s u l t s of t h i s a n a l y s i s are giv e n i n F i g u r e 21. 139 F i g u r e 20. I n d i r e c t immunofluorescent s t a i n i n g of mouse sperm by u s i n g mouse a n t i s e r a a g a i n s t MSA-63 f u s i o n p r o t e i n . Mouse sperm under v i s i b l e l i g h t and UV l i g h t are presented i n (A) and (B), r e s p e c t i v e l y (X 400). (The data were p u b l i s h e d i n L i u e t a l . , 1990) 140 141 F i g u r e 21. ELISA showing the b i n d i n g between mouse a n t i s e r a a g a i n s t MSA-63 f u s i o n p r o t e i n and mouse sperm homogenate coated on m i c r o w e l l s . Absorbance a t 450 nm was p l o t t e d as a f u n c t i o n of antiserum d i l u t i o n on a l o g s c a l e , where (•) denotes the mouse antiserum a g a i n s t f u s i o n MSA-63 p r o t e i n and (X) i s t h a t a g a i n s t E . c o l i h ost Y1089. (The data were p u b l i s h e d i n L i u e t a l . , 1990) 142 143 By u s i n g the mouse i n v i t r o f e r t i l i z a t i o n experiment, the mouse a n t i s e r a a g a i n s t recombinant f u s i o n p r o t e i n c o u l d s i g n i f i c a n t l y i n h i b i t the i n v i t r o f e r t i l i z a t i o n of mouse oocyte at a serum d i l u t i o n of 1:10 ( f e r t i l i z a t i o n r a t e of 37.7 % vs. 74.1 % f o r the c o n t r o l , P < 0.001). Supplemental d e t a i l s are presented i n Table 9. 144 Table 9. Inhibitory effect of mouse antisera raised against MSA-63 fusion protein on in vi t r o f e r t i l i z a t i o n of mouse oocytes (Liu et a l . , 1990)^ Immunogen Number of Ova examined Ova f e r t i l i z e d F e r t i l i z a t i o n P c assay (No.) (No.) rate (%) value Homogenate o f a 2 31 23 74.1 host Y1089 Recombinant0 2 61 23 37.7 <0.001 MSA-63 fusion protein aThe t i t r e of antiserum to MSA-63 fusion protein is 1:500 as determined by the indirect immunofluorescent assay. ^Recombinant MSA-63 fusion protein was partially purified from homogenate of bacteria lysogens by Sephacryl S-300 gel f i l t r a t i o n chromatography. Compared with the control group. P a r t IV. DEVELOPMENTAL STUDIES OF MSA-63 ANTIGEN A. CONSERVED NATURE OF SA-63 GENE AND ITS LOCATION ON HUMAN  CHROMOSOME To examine the d i v e r s i t y of SA-63 gene among d i f f e r e n t mammalian s p e c i e s , genomic DNA i s o l a t e d from dog, r a b b i t , r a t , guinea p i g , human, and mouse of both sexes were shown t o h y b r i d i z e with cDNA probe of MSA-63. R e s u l t s of t h i s Southern b l o t a n a l y s i s are shown i n F i g u r e 22. T h i s a n a l y s i s r e v e a l e d the conserved nature of SA-63 gene among d i f f e r e n t mammalian s p e c i e s . Moreover, the genomic DNA from e i t h e r sex ( F i g u r e 22, lanes 5-8) gave e s s e n t i a l l y the same p a t t e r n and i n t e n s i t y of h y b r i d i z a t i o n through t h i s a n a l y s i s . The l o c a t i o n of SA-6 3 gene on human chromosome was a l s o determined by u s i n g the b l o t of human-hamster somatic c e l l h y b r i d panel (from BIOS Co.). By u s i n g the MSA-63 cDNA i n s e r t with the s i z e of 1.2 Kb as a probe, the h y b r i d i z a t i o n s i g n a l was de t e c t e d on lanes 16 and 17 i n F i g u r e 23. P r e l i m i n a r y r e s u l t s of t h i s a n a l y s i s seemed t o suggest t h a t the SA-63 gene i s l o c a t e d on human chromosome 11 ac c o r d i n g t o the i n s t r u c t i o n manual from BIOS Co. 146 F i g u r e 22. Southern b l o t a n a l y s i s of SA-63 gene from d i f f e r e n t mammalian s p e c i e s . A: The Bgl I I d i g e s t e d genomic DNA on 0.8 % agarose g e l . B: The p a t t e r n of h y b r i d i z a t i o n with 3 z P - l a b e l e d M S A - 6 3 cDNA probe. DNA•specimens are from: lane 1, dog; lane 2, r a b b i t ; lane 3, r a t ; lane 4, guinea p i g ; lane 5, male mouse; lane 6, female mouse; lane 7, male human; lane 8, female human. Mixture of Lambda DNA d i g e s t e d by Hind I I I and <j>X174 RF DNA d i g e s t e d by Hae I I I i n Kb are shown on the l e f t as s i z e markers. (Work performed i n Dr. C h r i s Lau's l a b a t U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o ) (The data were p u b l i s h e d i n L i u e t a l . , 1 9 9 0 ) 147 A B 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 2 3 . 1 -0 .56 -148 F i g u r e 23. Southern b l o t a n a l y s i s o f SA-63 gene by u s i n g a b l o t of 3 P EcoR I d i g e s t e d human-hamster somatic c e l l h y b r i d p a n e l . The P labeled-MSA-63 cDNA c l o n e was used as h y b r i d i z a t i o n probe. Lanes 1 and 12 are the c o n t r o l of human DNA and lanes 11 and 22 are the c o n t r o l of hamster DNA. The human s p e c i f i c p o s i t i v e h y b r i d i z a t i o n s i g n a l s are i n d i c a t e d by arrows. (The b l o t was purchased from BIOS Co., New Haven, CT) 149 1 2 3 4 5 6 7 8 9 1 0 11 1 2 13 14 1 5 1 6 1 7 18 1 9 2 0 21 2 2 150 Bj, TISSUE-SPECIFIC EXPRESSION OF MSA-6 3 GENE By u s i n g the cDNA i n s e r t of cl o n e 12 as a probe, the ex p r e s s i o n o f MSA-63 gene was examined i n d i f f e r e n t mouse t i s s u e s by Northern b l o t a n a l y s i s . R e s u l t s of t h i s a n a l y s i s i n d i c a t e d t h a t t h i s cDNA probe c o u l d h y b r i d i z e t o a 1.5 Kb mRNA i n a d u l t mouse t e s t i s . However, no h y b r i d i z a t i o n of t h i s cDNA probe was observed with f e t a l t e s t i s , ovary o r any othe r mouse somatic t i s s u e s ( i . e . b r a i n ) from e i t h e r sex. R e s u l t s of t h i s a n a l y s i s are shown i n F i g u r e 24. C. EXPRESSION OF MSA-63 GENE IN DEVELOPING MOUSE TESTES RNA e x t r a c t e d from mouse t e s t e s of d i f f e r e n t ages (5 days t o 35 days old) were separated by agarose g e l e l e c t r o p h o r e s i s . Northern b l o t h y b r i d i z a t i o n w i t h 3 2 P - l a b e l e d cDNA probe of MSA-6 3 gene was performed. mRNA ex p r e s s i o n of MSA-63 was f i r s t d e t e c t e d on day 20 a f t e r b i r t h i n the deve l o p i n g mouse t e s t i s and reached a maximum l e v e l on day 35 a f t e r b i r t h . The h y b r i d i z a t i o n a n a l y s i s i s presented i n F i g u r e 25. The e x p r e s s i o n of MSA-63 p r o t e i n i n deve l o p i n g mouse t e s t i s 151 F i g u r e 24. Northern b l o t a n a l y s i s of MSA-63 gene e x p r e s s i o n i n d i f f e r e n t mouse t i s s u e s . The f i l t e r was h y b r i d i z e d w i t h 3 2 p -l a b e l e d MSA-63 cDNA c l o n e 12 and washed under hi g h s t r i n g e n t c o n d i t i o n s . The band was v i s u a l i z e d by autoradiography. Denatured PM2 DNA d i g e s t e d by Hind I I I are used as a s i z e marker i n Kb and i l l u s t r a t e d on the l e f t . RNA samples of mouse t i s s u e s a r e : lane 1, male b r a i n ; lane 2, female b r a i n ; lane 3, f e t a l male t e s t i s ; lane 4, f e t a l female ovary and lane 5, a d u l t t e s t i s , r e s p e c t i v e l y . (Work c a r r i e d out i n Dr. C h r i s Lau's l a b a t U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o ) (The data were p u b l i s h e d i n L i u e t a l . , 1990) 152 1 5.0-2.0-1.0-0.5-2 3 4 5 11.5 153 F i g u r e 25. Northern a n a l y s i s of RNA obtained from mouse t e s t e s a t v a r i o u s ages of development. Lane 1 t o 7 c o n t a i n s 20 ug of t o t a l RNA each from t e s t e s a t age of 5, 10, 15, 20, 25, 30, and 35 days old mice, r e s p e c t i v e l y . The f i l t e r was h y b r i d i z e d w i t h 3 2 P -labeled MSA-63 cDNA clo n e 12 and washed under high s t r i n g e n t c o n d i t i o n s . The s i z e markers, 28S and 18S ribosomal RNA are shown on the l e f t . 154 1 2 3 4 5 6 7 155 was a l s o s t u d i e d . Rabbit a n t i s e r a a g a i n s t MSA-63 served as the d e t e c t i n g probe i n a dot b l o t enzyme immunoassay f o r f o l l o w i n g the s p e c i f i c e x p r e s s i o n o f MSA-63 p r o t e i n i n t e s t i s from mice of d i f f e r e n t ages. As shown i n F i g u r e 26, the MSA-63 an t i g e n i n de v e l o p i n g mouse t e s t i s was not d e t e c t e d i n mouse t e s t i s u n t i l day 20 a f t e r the b i r t h . D. IMMUNOHISTOCHEMICAL STUDIES OF MSA-6 3 ANTIGEN IN MOUSE  TESTIS An immunohistochemical method was used t o analyze the ex p r e s s i o n of MSA-63 p r o t e i n i n dev e l o p i n g mouse t e s t i s . By us i n g i n d i r e c t immunofluorescent assay on the f r o z e n t e s t i s s e c t i o n s , i t c o u l d be c l e a r l y demonstrated t h a t MSA-63 was not de t e c t e d u n t i l day 25 a f t e r b i r t h . MSA-63 p r o t e i n was mainly l o c a l i z e d on the acrosomal r e g i o n bf the elongated spermatids. R e s u l t s of t h i s assay are presented i n F i g u r e 27. 156 F i g u r e 26. E x p r e s s i o n of MSA-63 a n t i g e n i n d e v e l o p i n g mouse t e s t e s . Dot 1 t o 8 c o n t a i n s 0.5 mg each supernatant of mouse t e s t e s homogenate a t the ages of day 5, 10, 15, 20, 25, 30, 35, and 60 r e s p e c t i v e l y . Dot 9 and 10 c o n t a i n 0.5 mg of mouse l i v e r homogenate supernatant and 12.5 ug BSA, r e s p e c t i v e l y . Immunoblot assay i s performed with (A) r a b b i t anti-MSA63 s e r a ; (B) r a b b i t anti-LDHX s e r a (C) normal r a b b i t serum. Column (D) was s t a i n e d by amido b l a c k f o r p r o t e i n q u a n t i t a t i v e c o n t r o l . D e t a i l s of t h i s assay procedure are giv e n i n the s e c t i o n of M a t e r i a l s and Methods. 157 1 2 3 4 5 6 7 8 9 10 A B " o c o o C D • • • • • • • • + • dav 5 10 15 20 25 30 35 60 L B 158 F i g u r e 27. Immunohistochemical a n a l y s i s of MSA-63 i n mouse t e s t e s a t v a r i o u s stage of development. The t e s t i c u l a r t i s s u e s e c t i o n s from A t o F are a t age of day 5, 10, 15, 20, 25, and 30 r e s p e c t i v e l y (X '400). 159 D : <*>, J r ? ..- - . • ,• 160 IV. DISCUSSION PART I . CHARACTERIZATION OF HS-63 REACTIVE SPERM ANTIGENS AND EVALUATION OF THEIR ROLES DURING FERTILIZATION PROCESSES The process of mammalian f e r t i l i z a t i o n i s a continuous and p r o g r e s s i v e event. Spermatozoa must undergo a sequence of programmed events i n c l u d i n g c a p a c i t a t i o n , acrosome r e a c t i o n and sperm-oocyte f u s i o n i n order t o complete the o v e r a l l f e r t i l i z a t i o n process ( F r a s e r and Ahuja, 1988; Wassarman, 1987a,b). To study such a complicated system, i t i s e s s e n t i a l t o i d e n t i f y those sperm p r o t e i n s which are .involved i n the c r u c i a l s t eps of the f e r t i l i z a t i o n p r o c e s s . Numerous e f f o r t s by ot h e r s have been made to i d e n t i f y those c r u c i a l sperm components mainly by u s i n g monoclonal sperm a n t i b o d i e s as the i n i t i a l t o o l s f o r the i d e n t i f i c a t i o n (Naz,et a l . , 1984a; O'Rand e t a l . , 1984; Gerton e t a l . , 1988; S a l i n g , 1986; Primakoff et a l . , 1988a). In our l a b o r a t o r y , HS-63 monoclonal antibody was i n i t i a l l y found t o r e a c t with the acrosome of human and mouse sperm ( F i g u r e 8 ) and i n h i b i t mouse i n v i t r o f e r t i l i z a t i o n (Lee e t a l . 1986; Menge e t a l . 1987; Anderson e t a l . , 1987; Table 2, 3, 4). I t was 161 t h e r e f o r e hypothesized t h a t the c o r r e s p o n d i n g sperm a n t i g e n (SA-63) i n the acrosome may be i n v o l v e d i n f e r t i l i z a t i o n p r o c e s s . HS-63 was shown to r e a c t with the acrosome of l i v e c a p a c i t a t e d sperm, but not wi t h the f r e s h u n c a p a c i t a t e d ones ( F i g u r e 3). Sperm c a p a c i t a t i o n i s a unique process r e q u i r e d f o r mammalian f e r t i l i z a t i o n . C e r t a i n m u l t i f a c e t e d changes of sperm s u r f a c e membrane and i n t r a c e l l u l a r components are r e q u i r e d d u r i n g the c a p a c i t a t i o n process ( F r a s e r , 1983, 1984). R e d i s t r i b u t i o n of intramembranous p a r t i c l e s and i n t r a c e l l u l a r c a l c i u m content i n the head and midpiece r e g i o n s of spermatozoa might be a s s o c i a t e d with sperm c a p a c i t a t i o n (Koehler and Gaddum-Rose, 1975; Bearer and F r i e n d , 1982). F o l l o w i n g the treatment of u n c a p a c i t a t e d sperm with e t h a n o l , h i g h s a l t (0.5 M KC1) or detergent such as T r i t o n X-100, SA-63 may be exposed to the sperm s u r f a c e and become a v a i l a b l e f o r antibody b i n d i n g . T h e r e f o r e , i t i s reasonable t o assume t h a t f o l l o w i n g c a p a c i t a t i o n , the SA-63 becomes exposed as the r e s u l t of a sperm s u r f a c e membrane change; T h i s o b s e r v a t i o n seemed t o suggest t h a t SA-63 may be. i n i t i a l l y l o c a l i z e d beneath the plasma membrane of the sperm acrosome, and becomes exposed f o l l o w i n g the c a p a c i t a t i o n p r o c e s s . 162 The sperm acrosome r e a c t i o n i n v o l v e s the f u s i o n of plasma and ou t e r acrosomal membranes and the r e l e a s e of h y d r o l y t i c enzymes, which i s s i m i l a r t o the process of e x o c y t o s i s ( R u s s e l l e t a l . , 1979; Wassarman, 1987a). Some of the sperm acrosomal p r o t e i n s r e s i d e i n the plasma and o u t e r acrosomal membrane were found to shed d u r i n g the acrosome r e a c t i o n (Morton, 1975; M e i z e l and M u k e r j i , 1975). However, p r o t e i n s a s s o c i a t e d with the i n n e r acrosomal membrane w i l l remain on the sperm s u r f a c e a f t e r completion of the acrosome r e a c t i o n ( i . e . a c r o s i n ) (Dunbar e t a l . , 1976). In view of the f a c t t h a t HS-63 c o u l d no longer r e a c t with the acrosome-reacted sperm, i t can be suggested t h a t SA-6 3 may l i e i n between the i n n e r acrosomal membrane and plasma or ou t e r acrosomal membrane. In summary, our experiments suggest t h a t SA-6 3 can be a u s e f u l marker f o r s t u d y i n g sperm c a p a c i t a t i o n and the acrosome r e a c t i o n . However, the exact l o c a t i o n of SA-63 a n t i g e n i n mammalian sperm remains t o be determined by e l e c t r o n microscopy. The i n h i b i t o r y e f f e c t of HS-63 on i n v i t r o f e r t i l i z a t i o n of mouse oocytes was found t o be dose-dependent (Table 2). When the c o n c e n t r a t i o n of HS-63 was d i l u t e d t o 0.04 mg/ml, there was nb 163 s i g n i f i c a n t i n h i b i t i o n o f f e r t i l i z a t i o n i n v i t r o . R e s u l t s of our experiments a l s o r e v e a l e d t h a t HS-63 had l i t t l e e f f e c t on sperm m o t i l i t y and sperm-zona b i n d i n g i n the mouse system. However, Wolf and h i s coworkers (Archibong e t a l . , 1991) observed t h a t HS-63 c o u l d s i g n i f i c a n t l y i n h i b i t human sperm-zona b i n d i n g i n a t y p i c a l human hemizona assay system. The di s c r e p a n c y c o u l d be due t o the d i f f e r e n t a f f i n i t y between HS-63 b i n d i n g t o human sperm and t h a t t o mouse sperm. In the human sperm p e n e t r a t i o n assay, HS-6 3 was found t o i n h i b i t not only the p e n e t r a t i o n r a t e but a l s o the mean number of human sperm att a c h e d t o zona-free hamster ova (Table 3). In a gi v e n sperm p e n e t r a t i o n assay, i t i s known t h a t the r e s u l t i n g f e r t i l i z a t i o n r a t e i s a s s o c i a t e d w i t h many p h y s i o l o g i c a l f a c t o r s such as the degree of sperm acrosome r e a c t i o n , sperm m o t i l i t y , a v i d i t y of b i n d i n g t o the egg s u r f a c e and e f f i c a c y of sperm-egg f u s i o n . (Fukuda e t a l . , 1989; van K o o i j e t a l . , 1986a,b; A i t k e n e t a l . , 1983; Yang e t a l . , 1988). The b i n d i n g of HS-63 t o c a p a c i t a t e d human sperm may bl o c k some of these f a c t o r s so t h a t the sperm p e n e t r a t i o n r a t e i s reduced. I t has been r e p o r t e d t h a t human sperm bound t o zona-free hamster ova were found t o be ac r o s o m e - r e a c t i v e . ( S i n g e r e t a l . , 1985). The number of bound human sperm on the s u r f a c e of zona-f r e e hamster ova may be r e l a t e d t o the degree of sperm acrosome r e a c t i o n (Singer e t a l . , 1985). I t i s p o s s i b l e t h a t the b i n d i n g of HS-63 t o c a p a c i t a t e d sperm c o u l d impede the r a t e of sperm acrosome r e a c t i o n , so t h a t the number of sperm atta c h e d t o zona-f r e e hamster ova was reduced s i g n i f i c a n t l y . The a b i l i t y of acrosome-reactive' sperm t o f e r t i l i z e eggs i s not w e l l known and c o u l d be s p e c i e s s p e c i f i c (Longo, 1987). In the case of mouse, onl y acrosome-intact sperm show the f e r t i l i z i n g c a p a c i t y . Acrosome-reacted mouse sperm can no longer b i n d t o the zona p e l l u c i d a of the ova ( S a l i n g e t a l . , 1979; Storey e t a l . , 1984; Wassarman, 1987a). However, i n humans and guinea p i g s , experimental evidence suggested t h a t the acrosome-r e a c t e d sperm might r e t a i n t h e i r a b i l i t y t o f e r t i l i z e the egg (Wolf, 1986; Yanagimachi, 1984). In view of the f a c t ' t h a t HS-63 only r e a c t s with the acrosome-intact c a p a c i t a t e d sperm, one would expect t h a t HS-63 can onl y i n h i b i t the i n i t i a l f e r t i l i z a t i o n s t eps p r i o r t o sperm p e n e t r a t i o n i n t o zona p e l l u c i d a and sperm-egg membrane f u s i o n . 165 In order to f u r t h e r e x p l o r e the p o s s i b l e i n h i b i t o r y mechanism of HS-63 on f e r t i l i z a t i o n , s e v e r a l i n v i t r o assays were performed i n c l u d i n g i t s e f f e c t on sperm-zona b i n d i n g and zona-induced acrosome r e a c t i o n . Our p r e l i m i n a r y experimental evidence showed t h a t although HS-63 has l i t t l e ' i n h i b i t o r y e f f e c t on mouse sperm-zona b i n d i n g , i t does s i g n i f i c a n t l y i n h i b i t zona-induced sperm acrosome r e a c t i o n . Judging from t h i s o b s e r v a t i o n , i t can be assumed t h a t the i n h i b i t o r y e f f e c t of HS-63 on f e r t i l i z a t i o n c o u l d r e s u l t from i t s i n t e r f e r e n c e with the steps of zona-induced acrosome r e a c t i o n p r i o r t o sperm p e n e t r a t i o n i n t o zona p e l l u c i d a . In view of the f a c t t h a t the mechanism of zona-induced acrosome r e a c t i o n i s c u r r e n t l y unknown (Roldan and H a r r i s o n , 1 9 9 0 ) , the exact stage a t which HS-63 i n t e r f e r e s with the acrosome r e a c t i o n remains t o be e l u c i d a t e d . A c c o r d i n g to p r e v i o u s s t u d i e s u s i n g c h l o r t e t r a c y c l i n e (CTC) f l u o r e s c e n t assay, the process of acrosome r e a c t i o n can be d i v i d e d i n t o t h r e e stages ( S a l i n g and Storey, 1979; Storey e t a l . , 1984). They are (1) F phase - sperm with i n t a c t acrosome, (2) S phase .- i n t e r m e d i a t e stage of sperm acrosome r e a c t i o n , and (3) AR phase - acrosome r e a c t e d sperm, r e s p e c t i v e l y . T h i s assay 166 was a l s o s u c c e s s f u l l y used t o study the i n h i b i t i o n of sperm antibody t o the acrosome r e a c t i o n ( S a l i n g e t a l . , 1986). M42 monoclonal antisperm antibody was found t o i n h i b i t zona-induced acrosome r e a c t i o n a t F phase and had no e f f e c t on ionophore-induced acrosome r e a c t i o n (Leyton e t a l . , 1989). I t was suggested t h a t M42 c o u l d a f f e c t c a l c i u m i n f l u x d u r i n g the acrosome r e a c t i o n and might not be i n v o l v e d i n membrane f u s i o n (Lyeton e t a l . , 1989). In view of t h i s r e p o r t , CTC s t a i n i n g assay may be used t o ev a l u a t e the e f f e c t o f HS-63 on sperm acrosome r e a c t i o n i n the f u t u r e . From the i n v i v o mating s t u d i e s with p a s s i v e immunization of female mice with HS-63, the antibody t i t r e of HS-63 i n mouse c i r c u l a t i n g s e r a c o u l d reach to 1:200,000 by u s i n g i n d i r e c t immunofluorescent assay. However, the l o c a l antibody t i t r e of HS-63 i n o v i d u c t f l u i d was estimated t o be 1:2,500. Judging from the antibody dose-dependent e f f e c t on the f e r t i l i z a t i o n of mouse oocytes i n v i t r o (Table 2), the l o c a l antibody t i t r e i n o v i d u c t f l u i d might be too low t o cause any i n h i b i t i o n on i n v i v o f e r t i l i z a t i o n . In v i v o mating experiments v i a p a s s i v e immunization of 167 monoclonal a n t i b o d i e s have been p r e v i o u s l y r e p o r t e d by S a l i n g and coworkers ( S a l i n g e t a l . , 1986). Two monoclonal a n t i b o d i e s , M29 (IgM s u b c l a s s ) and M42 (IgGl s u b c l a s s ) t h a t r e a c t e d s e p a r a t e l y with mouse sperm e q u a t o r i a l segment and acrosome were c l e a r l y demonstrated t o i n h i b i t mouse f e r t i l i z a t i o n i n v i v o f o l l o w i n g p a s s i v e immunization i n female mice. I t was observed t h a t i n c r e a s i n g the dose of these two a n t i b o d i e s l e d t o a s i g n i f i c a n t r e d u c t i o n i n f e r t i l i z a t i o n l e v e l . Since sperm antigens r e c o g n i z e d by HS-63 and M29 or M42 are ap p a r e n t l y d i f f e r e n t , the d i f f e r e n t i a l e f f e c t i v e n e s s between HS-63 and M29/M42 c o u l d be e x p l a i n e d . The s t a b i l i t y of the a n t i g e n i c determinant r e c o g n i z e d by HS-63 was s t u d i e d v i a treatments of sperm wi t h d e t e r g e n t s , p r o t e o l y t i c enzymes, g l y c o s i d a s e s , or carbohydrate modifying chemicals. Through such e x t e n s i v e a n a l y s e s , i t was concluded t h a t HS-63 r e a c t s with a pe p t i d e determinant of SA-63 which i s s t i l l immunoactive i n the presence of n o n i o n i c detergent. T h e r e f o r e , the recombinant f u s i o n p r o t e i n c a r r y i n g p a r t of SA-63 c o u l d s t i l l be r e c o g n i z e d by HS-63 ( F i g u r e 19). Meanwhile, through the molecular c l o n i n g of MSA-6 3 cDNA, the a n t i g e n i c determinants were t e n t a t i v e l y a s s i g n e d from the amino a c i d sequence of the deduced 168 p r o t e i n ( F i g u r e 16 and 17). However, h y d r o p h i l i c i t y i s o n l y one of the p r e d i c t o r s f o r a n t i g e n i c determinant. 169 P a r t I I . PURIFICATION AND CHARACTERIZATION OF MSA-63 By means of i n d i r e c t immunofluorescent assay, i t was c l e a r l y e s t a b l i s h e d t h a t MSA-63 a n t i g e n i n mouse t e s t i s e x i s t s as a membrane-associated s o l u b l e p r o t e i n . Thus, when the c o n v e n t i o n a l s o l u b l e p r o t e i n p u r i f i c a t i o n procedure was employed f o r p u r i f i c a t i o n of MSA-63 an t i g e n , the p u r i f i e d MSA-63 p r e c i p i t a t e d out d u r i n g s t o r a g e . T h i s i s perhaps due to hydrophobic aggregation of MSA-63 p r o t e i n i n the absence of detergent (Hjelmeland and Chrambach, 1984). A l s o , t h i s o b s e r v a t i o n f u r t h e r suggested t h a t MSA-63 an t i g e n i s a membrane-associated s o l u b l e p r o t e i n and e x i s t s as aggregates i n the n a t i v e s t a t e ( F i g u r e 8 ) . In view of t h i s problem, T r i t o n X-100 was i n t r o d u c e d i n the second p u r i f i c a t i o n procedure. In the presence of 0.1 % T r i t o n X-100, no p r e c i p i t a t i o n was found i n the s o l u t i o n c o n t a i n i n g p u r i f i e d MSA-63 s o l u t i o n . As shown i n Table 5, the detergent e x t r a c t i o n method r e s u l t e d i n a much h i g h e r p r o t e i n recovery of MSA-6 3 as compared t o t h a t o f the s o l u b l e e x t r a c t i o n method. I t was a l s o n o t i c e d t h a t i n the presence of T r i t o n X-100, the estimated immunoactivity of p u r i f i e d MSA-63 i s somewhat lower. 170 A f t e r 2D-gel e l e c t r o p h o r e s i s , p u r i f i e d MSA-63 d i s p l a y e d a number of p r o t e i n spots ( F i g u r e 6A and 6B). Some of the p r o t e i n s p o t s i n group 1, 2, and 4 were i d e n t i f i e d t o be sperm a c t i n fragments through amino a c i d sequence a n a l y s i s of the i s o l a t e d p e p t i d e s . The major a c t i n spots on a 2D-gel were found t o have molecular weights of 50 Kd (group 1) and 45 Kd (group 2) and p i s around 5. Presumably, they are major a c t i n fragments i n mouse sperm ( F l a h e r t y e t a l . , 1986). Rabbit a n t i s e r a a g a i n s t p r o t e i n spots i n group 1 e x h i b i t e d h i g h c r o s s - r e a c t i v i t y t o - a c t i n . However, those r a i s e d a g a i n s t p r o t e i n spots i n group 2 showed l i t t l e c r o s s - r e a c t i v i t y t o ;..-actin. Through the a n a l y s i s o f amino a c i d sequence homology of p e p t i d e s i s o l a t e d from p r o t e i n spots i n group 1, 2, and 4, i t was concluded t h a t they are e i t h e r a c t i n -r e l a t e d p r o t e i n s or t h e i r d e g r a d a t i o n products. By u s i n g Western b l o t a n a l y s i s , a n t i s e r a r a i s e d a g a i n s t p r o t e i n spots i n group 3 (MSA-63) c o u l d c r o s s - r e a c t w i t h a l l the p r o t e i n spots on a 2D-gel. However, a n t i s e r a a g a i n s t p r o t e i n spots of group 1 and 2 ( a c t i n s ) c o u l d not r e a c t w i t h MSA-63 i n group 3. I t i s p o s s i b l e t h a t p r o t e i n spots i n group 3 which were used to r a i s e anti-MSA-63 s e r a may be contaminated with a c t i n - r e l a t e d 171 p r o t e i n fragments. Through g e l f i l t r a t i o n a n a l y s i s , i t was c l e a r l y demonstrated t h a t MSA-63 e x i s t s as aggregates i n the n a t i v e form w i t h a molecular s i z e r a n g i n g from 200 to 300 KD ( F i g u r e 8 ) . The aggregated form of MSA-63 c o u l d r e s u l t from e i t h e r s e l f a s s o c i a t i o n or complex formation w i t h a c t i n - r e l a t e d p r o t e i n s . I t i s i n t r i g u i n g t o f i n d t h a t a c t i n - r e l a t e d p r o t e i n s or fragments are t i g h t l y a s s o c i a t e d w i t h MSA-63 p r o t e i n . The i n t e r a c t i o n between MSA-63 and a c t i n (Sigma A2522) has a l s o been i n v e s t i g a t e d ( F i g u r e 9 ) . I t i s assumed t h a t changes of mole c u l a r d i s t r i b u t i o n of <?c-actin may be due t o the i n t e r a c t i o n between MSA-6 3 and a c t i n o r de g r a d a t i o n caused by MSA-63. The presence of a c t i n i n mammalian spermatozoa has been r e p o r t e d (Camatnini e t a l . , 1986; 1987; Casale e t a l . , 1988; F l a h e r t y e t a l . , 1983, 1986; Olson and Winfery, 1985). However, the l o c a t i o n s of a c t i n i n sperm vary with d i f f e r e n t mammalian s p e c i e s ( F l a h e r t y , 1986). The p r e c i s e f u n c t i o n a l r o l e of a c t i n i n sperm i s c u r r e n t l y unknown (Vogl, 1989). In mouse sperm, F - a c t i n was found on the s u r f a c e of 172 postacrosomal r e g i o n ( C l a r k e and ' Yanagimachi, 1978), midpiece and a n t e r i o r p r i n c i p a l p i e c e ( F l a h e r t y , 1986). R e s u l t s of our study may suggest t h a t , a c t i n s are a s s o c i a t e d with MSA-63 p r o t e i n i n the acrosomal r e g i o n and may be i n v o l v e d i n sperm acrosome r e a c t i o n . The p r o t e o l y t i c a c t i v i t y i n the p u r i f i e d MSA-63 p r e p a r a t i o n was s t u d i e d . When the p u r i f i e d MSA-63 was incubated a t 37°C f o r 5 days and two weeks a t n e u t r a l pH, the p r o t e i n spots i n group 3 r e v e a l e d no s i g n i f i c a n t changes. However, groups 1 and 2 p r o t e i n spots were found t o disappear when analyzed on a 2D-gel ( F i g u r e 15C). On the other hand, new p r o t e i n spots with molecular s i z e s r a n g i n g from 10 t o 20 Kd appeared on 2D-gel a f t e r long i n c u b a t i o n . The o r i g i n and the nature of the p r o t e o l y t i c a c t i v i t y i n the p u r i f i e d MSA-63 p r e p a r a t i o n remain t o be e l u c i d a t e d . MSA-63 p r o t e i n (group 3 on a 2D-gel) showed s e r i a l of p r o t e i n spots with s i m i l a r p i (about 4.2) but d i f f e r e n t molecular s i z e s r a n g i n g from 24 t o 45 Kd. Ac c o r d i n g t o the amino a c i d sequence deduced from f u l l l e n g t h cDNA i n s e r t , the molecular weight o f MSA-63 should be 27,948 d a l t o n . The treatment of O- and N-g l y c o s i d a s e or redu c i n g reagent f a i l e d t o change the 173 m i c r o h e t e r o g e n i c i t y of MSA-63 p r o t e i n on 2D-gel ( F i g u r e 10). Hence, one can assume t h a t the presence of carbohydrate moiety and d i s u l f i d e l i n k a g e may not be the cause o f such m i c r o h e t e r o g e n i c i t y i n MSA-63. I t i s t h e r e f o r e proposed the molecular s i z e h e t e r o g e n e i t y o f MSA-63 p r o t e i n may r e s u l t from the c r o s s - l i n k i n g or c o v a l e n t aggregations o f MSA-6 3 or i t s a s s o c i a t i o n w i t h other molecules such as a c t i n s . F u r t h e r t r y p t i c a n a l y s i s of MSA-63 p r o t e i n (group 3) should enable us t o s o l v e t h i s q u e s t i o n . 174 P a r t I I I . CLONING AND CHARACTERIZATION OF MSA-63 cDNA The cDNA encoding MSA-63 p r o t e i n was s u c c e s s f u l l y c l o n e d by u s i n g immunoscreening of a mouse t e s t i s cDNA l i b r a r y c o n s t r u c t e d i n the lambda g t 11 e x p r e s s i o n v e c t o r (Young and Davis, 1983). G e n e r a l l y most, cDNA i n s e r t s i s o l a t e d from the immuno-d e t e c t a b l e cDNA c l o n e s are not f u l l l e n g t h (Synder e t a l . , 1 9 8 7 ) . P o l y c l o n a l a n t i s e r a have the advantage of r e c o g n i z i n g m u l t i p l e e p i t o p e s of a g i v e n p r o t e i n . A monoclonal antibody can on l y r e c o g n i z e a s i n g l e e p i t o p e and i s t h e r e f o r e of l i m i t e d use f o r immunoscreening. Thus, p o l y c l o n a l a n t i s e r a are u s u a l l y recommended as probes f o r immunoscreening (Huynh e t a l . , 1985). Using t h i s technique, the cDNA c l o n e s of MSA-63 an t i g e n were i s o l a t e d from a mouse t e s t i s cDNA l i b r a r y by immunoscreening u s i n g r a b b i t antiserum a g a i n s t MSA-63. Through immunoscreening of 6 x 1 0 5 bacteriophages from mouse t e s t i s cDNA l i b r a r y , more than 60 p o s i t i v e c l o n e s were i d e n t i f i e d ( F i g u r e 12). The most d i f f i c u l t p a r t of cDNA i s o l a t i o n by immunoscreening i s t o determine whether the immunoreactive c l o n e s are d e r i v e d 175 from the gene of i n t e r e s t . Southern b l o t a n a l y s i s was u s u a l l y employed t o determine i f the i n s e r t s of the p o s i t i v e cDNA c l o n e s can c r o s s - h y b r i d i z e with each o t h e r . F o l l o w i n g h y b r i d i z a t i o n , o n l y those w i t h a hi g h degree of homology showed p o s i t i v e s i g n a l s under h i g h l y s t r i n g e n t wash c o n d i t i o n s . As a r e s u l t , f o u r p o s i t i v e cDNA c l o n e s were i s o l a t e d (see F i g u r e 13). From the r e s t r i c t i o n mapping a n a l y s i s , they were shown to be d e r i v e d from a s i n g l e s t r u c t u r a l gene. Experiments were performed t o determine i f the i s o l a t e d cDNA clo n e s express MSA-63 p r o t e i n . The recombinant f u s i o n p r o t e i n prepared from the p o s i t i v e cDNA c l o n e s c o u l d be r e c o g n i z e d by anti-MSA-63 s e r a i n the Western b l o t assay ( F i g u r e 18). C e l l l y s a t e s of lysogen which produced the recombinant f u s i o n p r o t e i n were ab l e t o i n h i b i t HS-6 3 b i n d i n g t o sperm (see F i g u r e 19). Th e r e f o r e , i t can be suggested t h a t HS-63 r e a c t i v e e p i tope e x i s t s i n the recombinant f u s i o n p r o t e i n produced from E c o i i . R e s u l t s of t h i s experiment f u r t h e r confirmed t h a t the HS-63 s p e c i f i c e p i t o p e i s a pept i d e and not the carbohydrate moiety. The PC\GENE computer program was used t o analyze the h y d r o p h o b i c / h y d r o p h i l i c nature of cDNA deduced amino a c i d 176 sequence of MSA-6 3 p r o t e i n . A hydrophobic amino terminus was i d e n t i f i e d i n the deduced amino a c i d sequence, p o s s i b l y being an e u k a r y o t i c s e c r e t o r y s i g n a l (von H e i j n e , 1986). T h e r e f o r e , MSA-63 p r o t e i n may be t r a n s p o r t e d a c r o s s the membrane as a s e c r e t o r y p r o t e i n a f t e r i t s s y n t h e s i s . Due t o the l a c k o f membrane-spanning segment i n the e n t i r e amino a c i d sequence, MSA-63 i s co n s i d e r e d as a p e r i p h e r a l p r o t e i n , but not a membrane i n t e g r a l p r o t e i n ( K l e i n e t a l . , 1985). T h i s p r e d i c t i o n i s i n agreement with our e a r l i e r o b s e r v a t i o n t h a t MSA-63 i s a membrane-associated s o l u b l e p r o t e i n . Other c y t o s k e l e t a l p r o t e i n components such as a c t i n s may be r e q u i r e d t o anchor MSA-6 3. The s i z e of mRNA i d e n t i f i e d from mouse t e s t i s was c l o s e t o 1.5 Kb (F i g u r e 24). However, the i s o l a t e d cDNA cl o n e s had a s i z e of o n l y 1.2 Kb ( F i g u r e 16). I t i s p o s s i b l e t h a t the p o l y A t a i l of the mRNA may be more than 200 bp. Fu r t h e r study by u s i n g the method of mRNA primer e x t e n s i o n may be r e q u i r e d t o i d e n t i f y the s i z e of f u l l l e n g t h MSA-63 cDNA. U s u a l l y , the p o l y a d e n y l a t i o n i n e u k a r y o t i c mRNA i s r e q u i r e d t o s t a b i l i z e the mRNA f o r long term e x p r e s s i o n (Burton e t a l . , 1983; Belasco e t a l . , 1 9 8 5 ) . However, an mRNA consensus 177 d e g r a d a t i o n sequence (AUUUA) was found i n the 3' noncoding r e g i o n . The presence of t h i s sequence seems t o promote mRNA decay (Shaw and Kamah, 1986; Caput e t a l . , 1986). T r a n s i e n t l y - e x p r e s s e d mRNAs, such as those f o r oncogenes, lymphokines and i n t e r f e r o n s , a l s o c a r r y such a segment i n t h e i r 3' noncoding r e g i o n (Wilson and Treisman, 1988; Jones and Cole, 1988). T h e r e f o r e , the e x p r e s s i o n of MSA-6 3 gene may a l s o be r e g u l a t e d by t h i s segment d u r i n g spermatogenesis. The computer search from the data base of the PC\GENE program ( r e l e a s e 6.25, J u l y 10, 1990) r e v e a l e d t h a t t h e r e was no s i g n i f i c a n t homology of MSA-6 3 with any o t h e r known n u c l e o t i d e s and p r o t e i n s . However, 60 t o 70 % homology was observed between MSA-6 3 cDNA sequence and t h a t of a human sperm acrosomal an t i g e n , SP-10 (Wright e t a l . , 1990). In c o n t r a s t , SP-10 a n t i g e n was not conserved i n a l l s p e c i e s . Sperm from s e v e r a l mammalian s p e c i e s i n c l u d i n g r a b b i t , b u l l , r a t , guinea p i g , and c a t do not express any a n t i g e n which i s immunologically or s t r u c t u r a l l y r e l a t e d t o SP-10 a n t i g e n (Herr e t a l . , 1990a,b). MHS-10, the monoclonal antibody by which SP-10 a n t i g e n was i d e n t i f i e d , was shown p r e v i o u s l y t o i n h i b i t f e r t i l i z a t i o n by the WHO workshop (Anderson et a l . , 1987). The s t r u c t u r a l and f u n c t i o n a l r e l a t i o n s h i p between 178 MSA-63 and SP-10 a n t i g e n i n sperm acrosome remain t o be e x p l o r e d i n the near f u t u r e . 179 P a r t IV. DEVELOPMENTAL STUDY OF MSA-6 3 ANTIGEN A p r e v i o u s study showed t h a t HS-63 monoclonal antibody i s s p e c i e s conserved and sperm s p e c i f i c (Anderson e t a l . , 1987). At the molecular l e v e l , by Southern b l o t a n a l y s i s u s i n g MSA-63 cDNA as a probe, the SA-6 3 gene was found t o be conserved among d i f f e r e n t s p e c i e s i n both sexes ( F i g u r e 22). In human, the l o c a t i o n of the SA-63 gene was suggested t o be on chromosome 11 (F i g u r e 23). T h e r e f o r e , the SA-63 gene i s l o c a l i z e d on an autosome, and not on the sex chromosome. R e s u l t s o f these molecular analyses are c o n s i s t e n t with those of immunological a n a l y s i s by . the WHO workshop r e g a r d i n g the broad s p e c i e s c r o s s - r e a c t i v i t y of HS-63 monoclonal antibody (Anderson e t a l , 1987). R e s u l t s of the Northern b l o t a n a l y s i s showed t h a t the mRNA of MSA-63 was only expressed i n a d u l t t e s t i s but not i n f e t a l t e s t i s or any oth e r somatic t i s s u e s ( F i g u r e 24). In the de v e l o p i n g mouse t e s t i s , spermatogenesis begins on day 5 p o s t n a t a l l y , when the p r i m i t i v e type A spermatogonia begin t o p r o l i f e r a t e ( B e l l v e ' e t a l . , 1977). On day 9, type B spermatogonia pass through a m i t o t i c d i v i s i o n t o y i e l d p r e l e p t o t e n e spermatocytes. By day 10, leptotene.spermatocytes appear and q u i c k l y d i f f e r e n t i a t e t o form 180 zygotene spermatocytes. By day 14, pachytene spermatocytes are formed. Around day 18 of development, the f i r s t m e i o t i c d i v i s i o n occurs t o form secondary spermatocytes ( B e l l v e ' , 1979). On day 20, a s i g n i f i c a n t number of pachytene spermatocytes i n mouse t e s t i s are ready t o en t e r the round spermatid stage (Hecht e t a l . , 1985). The t r a n s c r i p t i o n of MSA-63 was f i r s t d e t e c t e d i n mouse t e s t i s on day 20 a f t e r b i r t h and the maximum e x p r e s s i o n was found on day 35 ( F i g u r e 25). T h e r e f o r e , the nature of gene e x p r e s s i o n of MSA-6 3 appears t o be p o s t m e i o t i c . At the p r o t e i n l e v e l , the immunoactivity of MSA-63 a n t i g e n was f i r s t d e t e c t e d i n t e s t i s of 20 day o l d mice ( F i g u r e 26). T h i s o b s e r v a t i o n suggests t h a t t r a n s c r i p t i o n and t r a n s l a t i o n of MSA-6 3 gene are i n i t i a t e d s i m u l t a n e o u s l y i n the de v e l o p i n g mouse t e s t i s . Sperm s p e c i f i c genes are v a l u a b l e f o r the study of the r e g u l a t i o n of gene e x p r e s s i o n d u r i n g spermatogenesis (Kleene e t a l . , 1984; Hecht e t a l . , 1985; Waters e t a l . , 1985; V i l l a s a n t e e t a l . , 1986; Mutter and Wolgemith, 1987). L a c t a t e dehydrogenase-X ( L D H-X), a sperm s p e c i f i c isozyme, i s expressed i n the h a p l o i d germ c e l l (Hintz and Goldberg, 1977; Wheat e t a l . , 1977; 181 M e i s t r i c h e t a l . , 1977; Wieben, 1981; Tanaka and Fujimoto, 1986). T r a n s c r i p t i o n and t r a n s l a t i o n of the LDH-X gene are i n i t i a t e d a t the pachytene stage of germ c e l l d i f f e r e n t i a t i o n (Jen e t a l . , 1990), the stage of which i s c o n s i d e r e d p r e m e i o t i c . Protamine, a t e s t i s - s p e c i f i c h i s t o n e , i s found t o be expressed i n the d i p l o i d c e l l d u r i n g m e i o t i c prophase (Hecht e t a l . , 1985). However, the message of protamine i s s t o r e d without t r a n s l a t i o n u n t i l the stage of h a p l o i d spermatids. Phosphoglycerate kinase-2 (PGK-2) i s a sperm s p e c i f i c isozyme which i s i n v o l v e d i n the g l y c o l y t i c metabolism of spermatozoa (Gold e t a l . , 1983). The p o s t m e i o t i c e x p r e s s i o n of PGK-2 i s a l s o s u b j e c t t o s t r o n g t r a n s l a t i o n a l c o n t r o l . As w e l l as protamine, the mRNA of PGK-2 i s s t o r e d f o r many days b e f o r e being t r a n s l a t e d i n the round spermatid stages of spermatogenesis (Gold e t a l . , 1983). S e v e r a l proto-oncogenes are a l s o found t o be expressed d u r i n g spermatogenesis ( i . e . c-mos, c - a b l and N - r a s ) . The e x p r e s s i o n of these proto-oncogenes may be a s s o c i a t e d with the c o n t r o l of sperm p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i n the t e s t i s ( W i l l i s o n and 182 The t i m i n g of MSA-6 3 e x p r e s s i o n i s around the onset of spermiogenesis. At t h i s moment, mo r p h o l o g i c a l changes i n round spermatids occur c o n c o m i t a n t l y w i t h a l t e r a t i o n s i n the complement of c o n s t i t u e n t p r o t e i n s ( B o i t a n i e t a l . , 1980; M i l l e t t e and Moulding, 1981; St e r n e t a l . , 1983). Meanwhile, acrosome formation i s i n i t i a t e d immediately a f t e r the completion o f m e i o s i s and i n t i m a t e l y i n v o l v e s the G o l g i apparatus i n e a r l y spermatids (Clermont and Tang, 1985). From our study on the gene e x p r e s s i o n of MSA-63,- i t can be concluded t h a t MSA-63 i s produced at the i n i t i a l stages of acrosome fo r m a t i o n . F a c t o r s t h a t r e g u l a t e the MSA-6 3 gene e x p r e s s i o n i n spermatogenesis are unknown. In t e s t i s , sperm c e l l s are i n i n t i m a t e c o n t a c t with S e r t o l i c e l l s d u r i n g a l l phases of spermatogenesis, which i n c l u d e s m e i o t i c p r o l i f e r a t i o n , m e i o t i c d i v i s i o n , and d i f f e r e n t i a t i o n ( R u s s e l l , 1980). I t i s b e l i e v e d t h a t S e r t o l i c e l l s may p l a y an important r e g u l a t o r y r o l e i n spermatogenesis ( T i n d a l l e t a l . , 1985). Hence, the e x p r e s s i o n of MSA-63 antig e n might be r e g u l a t e d d i r e c t l y o r i n d i r e c t l y by the S e r t o l i c e l l s . T h i s hypothesis remains t o be confirmed. 183 V. CONCLUSION AND FUTURE RESEARCH DIRECTION During the l a s t two decades, numerous b i o t e c h n i g u e s have been developed f o r a p p l i c a t i o n s i n b i o m e d i c a l r e s e a r c h . They have g r e a t l y r e v o l u t i o n i z e d the approaches by which s c i e n t i s t s s o l v e the important problems i n r e p r o d u c t i v e b i o l o g y . In t h i s study, hybridoma, p r o t e i n chemistry, and recombinant DNA technology r e p r e s e n t the most important t o o l s i n dealing- with problems r e l a t e d t o s t r u c t u r a l and f u n c t i o n a l r o l e s of s p e c i f i c sperm antigens d u r i n g the f e r t i l i z a t i o n . Without adequate a p p l i c a t i o n s of these techniques, i t would have been i m p o s s i b l e t o o b t a i n the i n f o r m a t i o n presented i n t h i s t h e s i s . S y s t e m a t i c a l l y , w i t h the a p p l i c a t i o n of hybridoma technology, numerous monoclonal a n t i b o d i e s t o sperm were generated i n our l a b o r a t o r y . Based on t h e i r r e s p e c t i v e sperm s p e c i f i c i t y and i n h i b i t o r y e f f e c t s on sperm f u n c t i o n s and f e r t i l i z a t i o n , HS-63 monoclonal antibody was among those s e l e c t e d . By means of immunoaffinity chromatography u s i n g t h i s antibody as the l i g a n d , s p e c i f i c mouse sperm a n t i g e n (MSA-63) r e c o g n i z e d by HS-63 was p u r i f i e d and c h a r a c t e r i z e d b i o c h e m i c a l l y and immunologically. 184 Furthermore, through the immunoscreening of a mouse t e s t i s cDNA l i b r a r y w i t h a n t i s e r a a g a i n s t MSA-63 as the probe, cDNA c l o n e s e x p r e s s i n g t h i s sperm p r o t e i n were i s o l a t e d . The molecular c l o n i n g work enabled us t o determine mRNA and p r o t e i n s t r u c t u r e s . The i s o l a t e d s p e c i f i c cDNA fragments were a l s o used as probes t o study the e x p r e s s i o n of t h i s sperm a n t i g e n d u r i n g spermatogenesis. Moreover, mass-production of s p e c i f i c sperm anti g e n s can a l s o be achieved v i a a p p l i c a t i o n s of recombinant DNA technology f o r the development o f a sperm a n t i g e n based immunocontraceptive v a c c i n e . Although much i n f o r m a t i o n has been obtained r e g a r d i n g the mRNA and p r o t e i n s t r u c t u r e of t h i s sperm s p e c i f i c acrosomal an t i g e n (SA-63), more s t u d i e s need t o be c a r r i e d out f o r the f u l l u nderstanding of i t s s t r u c t u r e and f u n c t i o n . In order t o determine the o r g a n i z a t i o n a l and s t r u c t u r a l f e a t u r e s of the genes encoding the SA-63 p r o t e i n , the e n t i r e SA-63 gene w i l l be cloned and sequenced. I t s exons, i n t r o n s and r e g u l a t o r y elements can be i n v e s t i g a t e d . Furthermore, the r e g u l a t i o n of the SA-63 gene e x p r e s s i o n can be eva l u a t e d by t r a n s f e c t i o n s t u d i e s by i n t r o d u c t i n g the e n t i r e gene or p u t a t i v e 185 r e g u l a t o r y r e g i o n s i n t o t i s s u e c u l t u r e c e l l s or a t r a n s g e n i c animal system so t h a t s p e c i f i c e x p r e s s i o n of SA-63 gene can be s t u d i e d i n response t o t r a n s a c t i n g r e g u l a t o r y f a c t o r s such as growth f a c t o r s and hormones. Moreover, the a v a i l a b i l i t y of HS-63 monoclonal antibody and a the s p e c i f i c cDNA probe f o r SA-63 w i l l enable us t o study the s u b c e l l u l a r l o c a t i o n of p r o t e i n and mRNA i n v a r i o u s d i f f e r e n t i a t e d spermatogenic c e l l s i n the t e s t i s and i n mature spermatozoa d u r i n g epididymal t r a n s i t . P a r t i c u l a r a t t e n t i o n w i l l be p a i d t o i d e n t i f y how SA-63 i s a s s o c i a t e d w i t h c y t o s k e l e t a l p r o t e i n s such as a c t i n s . In view of the p r o t e i n chemistry, i t would be i n t e r e s t i n g t o determine how the p r o t e i n expressed by the SA-63 gene can e x h i b i t such a broad molecular s i z e d i s t r i b u t i o n . The molecular mechanisms of such a wide-spread m i c r o h e t e r o g e n e i t y of SA-6 3 p r o t e i n and i t s s t r u c t u r a l and f u n c t i o n a l i m p l i c a t i o n s are an important s u b j e c t f o r f u r t h e r i n v e s t i g a t i o n s . Although the r e s u l t s of our p r e l i m i n a r y s t u d i e s r e v e a l e d t h a t M S A - 6 3 i s i n e f f e c t i v e as an immunocontraceptive v a c c i n e i n 186 mouse, the c o n t r a c e p t i v e e f f i c a c y of SA-63 i n primate s p e c i e s remains t o be e x p l o r e d . Because of s i g n i f i c a n t anatomical and p h y s i o l o g i c a l d i f f e r e n c e s i n the r e p r o d u c t i v e systems between rodents and primates, one would expect d i f f e r e n c e s i n c o n t r a c e p t i v e e f f i c a c y i n these two mammalian s p e c i e s . A c c o r d i n g to r e c e n t s t u d i e s by Wolf and h i s coworkers i n Oregon Regional Primate Center (Archibong e t a l . , 1991), a s t r o n g i n h i b i t i o n of HS-63 and r a b b i t anti-MSA-63 s e r a on sperm-zona b i n d i n g was found i n a human hemi-zona assay system. The r e s u l t of t h i s study suggests t h a t SA-6 3 may be a s u i t a b l e c andidate f o r immunocontraceptive v a c c i n e i n humans. A c t i v e immunizations w i t h p u r i f i e d SA-6 3 from rhesus monkey t e s t e s i n rhesus monkeys are a l r e a d y being underway a t the Oregon Regional Primate Center. 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(1986) Immunological approaches t o c o n t r a c e p t i o n : the need, b a s i c premise, and overview, i n : Immunological Approaches t o C o n t r a c e p t i o n and Promotion of F e r t i l i t y . Talwar, G.P. (ed.) Plenum Press, New York. pp. 17 Tanaka, S. and Fujimoto, H. (1986) A p o s t m e i o t i c a l l y expressed clone encodes l a c t a t e dehydrogenase isozyme X. Biochem. Biophys. Res. Commun. 136, 760 T e s a r i k , J . , T r a v n i k , P., Mecova, L. and P i l k a , L. (1985) B i n d i n g of antisperm i s o a n t i b o d i e s from a woman a t r i s k f o r immunological i n f e r t i l i t y t o i n t r a a c r o s o m a l components of donor spermatozoa. F e r t i l . S t e r i l . 44, 138 Teuscher, C., Wild, G. C. and Tung, K. S. K. (1982). Immunochemical a n a l y s i s of guinea p i g sperm autoantigens. B i o l . Reprod. 26, 218. Teuscher, C., Wild, G. C. and Tung, K. S. K. (1983). Acrosomal autoantigens of guinea p i g sperm. J . Immunol. 130, 317. Thomas, P. S. (1980) H y b r i d i z a t i o n of denatured RNA and s m a l l DNA fragments t r a n s f e r r e d t o n i t r o c e l l u l o s e . Proc. N a t l . Acad. S c i . (USA) 77, 5201. T i n d a l l , D.J., Rowley, D.R., Muethy, L., L i p s h u l t z , L . I . and Chang, C H . (1985) S t r u c t u r e and Bio c h e m i s t r y of the S e r t o l i c e l l . I n t . Rev. Cyto. 94, 127 Towbin, H., S r a c h e l i n , T., Gordon, J.A. (1979) E l e c t r o p h o r e t i c t r a n s f e r o f p r o t e i n from p o l y a c r y l a m i d e g e l s t o n i t r o c e l l u l o s e s h eets: Procedure and some a p p l i c a t i o n s . Proc. N a t l . Acad. S c i . (USA) 76, 4350 Tung, K.S..K. (1980). Immunological Aspects of I n f e r t i l i t y and F e r t i l i t y R e g u l a t i o n . In Dhinsda, D.S. and Schumacher, G.F.B. (eds.) E l s e v i e r Press, North H o l l a n d , pp. 3 3 van K o o i j , R.J., B a l e r n a , M., R o a t t i , A., Campana, A. (1986a) Oocyte p e n e t r a t i o n and acrosome r e a c t i o n s o f human spermatozoa. I: I n f l u e n c e of i n c u b a t i o n time and medium composition. A n d r o l o g i a 18, 152 van K o o i j , R.J., Balerna, M., R o a t t i , A., Campana, A. (1986b) Oocyte p e n e t r a t i o n and acrosome r e a c t i o n s of human spermatozoa. I I : C o r r e l a t i o n w i t h other seminal pamameters. A n d r o l o g i a 18, 503 199 Varmus, H.E., Q u i n t r e l l , N., and O r t i z , S. (1981) R e t r o v i r u s e s as mutagens : i n s e r t i o n and e x c i s i o n of a nontransforming p r o v i r u s a f t e r e x p r e s s i o n of a r e s i d e n t t r a n s f o r m i n g p r o v i r u s . C e l l 25, 23. V i l l a s a n t e , A., Wang, D., Dobner, P., Dolpph, P., Lewis, S.A. and Cowan, N.J. (1986) S i x mouse a l p h a - t u b u l i n mRNAs encode f i v e d i s t i n c t i s o t y p e s : T e s t i s - s p e c i f i c e x p r e s s i o n of two s i s t e r genes. Mol. C e l l B i o l . 6, 2409 Vogl, A.W. (1989) D i s t r i b u t i o n and f u n c t i o n of o r g a n i z e d c o n c e n t r a t i o n s of a c t i n f i l a m e n t s i n mammalian spermatogenic c e l l s and S e r t o l i c e l l s . I n t . Rev. Cyto. 119, 1 von H e i j n e , G. (1986) A new method f o r p r e d i c t i n g s i g n a l sequence cleavage s i t e s . N u c l e i c A c i d s Res. 14, 4683 Wassarman, PM (1987a) The b i o l o g y and chemistry of f e r t i l i z a t i o n . S c i e n ce 235, 553 Wassarman, PM (1987b) E a r l y events i n mammalian f e r t i l i z a t i o n . Ann. Rev. C e l l B i o l . 3, 109 Waters, S.H., D i s t e l , R.J. Hecht, N.B. (1985) Mouse t e s t e s c o n t a i n two s i z e c l a s s e s of a c t i n mRNA t h a t are d i f f e r e n t i a l l y expressed d u r i n g spermatogenesis. Mol. C e l l B i o l . 5, 1649 Welch, J.E. and O'Rand, M.G. (1985) I d e n t i f i c a t i o n and d i s t r i b u t i o n of a c t i n i n spermatogenic c e l i s and spermatozoa of the r a b b i t . Dev. B i o l . 109, 411 Wheat, T.E., H i n t z , M., Goldberg, E. and M a r g o l i a s h , E. (1977) A n a l y s i s of s t a g e - s p e c i f i c m u l t i p l e forms of l a c t a t e dehydrogenase and of cytochrome c d u r i n g spermatogenesis i n the mouse. D i f f e r e n t i a t i o n 9, 37 Wieben, E.D. (1981) R e g u l a t i o n of the s y n t h e s i s of l a c t a t e dehydrogenase-X d u r i n g spermatogenesis i n the mouse. J . C e l l B i o l . 88, 492 W i l l i s o n , K. and Ashworth, A. (1987) Mammalian spermatogenic gene e x p r e s s i o n . Trends Genet. 3, 351 Wilson, T. and Treisman, R. (1988) Revmoval of p o l y (A) and consequent f a c i l i t a t e d by 3' A U - r i c h sequences. Nature 336, 396 Wolf, D.P., S o k o l o s k i , J.E., Dandekar, P. and B e c h t o l , K.B. (1983) C h a r a c t e r i z a t i o n of human sperm s u r f a c e antigens w i t h monoclonal a n t i b o d i e s . B i o l . Reprod. 29, 713 Wolf, D.P., B o l d t , J . , Byrd, W., B e c h t o l , K.B. (1985) Acrosomal s t a t u s e v a l u a t i o n i n human e j a c u l a t e d sperm with monoclonal a n t i b o d i e s . B i o l . Reprod. 32, 1157 Wolf, D.P'. (1986) Research f r o n t i e r s i n human i n v i t r o 200 f e r t i l i z a t i o n . Adv. Exp. Med. B i o l . 207, 429 Wright, R.M., John, E., K l o t z , K., F l i c k i n g e r , C.J. and Herr, J . C. (1990) C l o n i n g and sequencing of cDNAs coding f o r the human in t r a - a c r o s o m a l a n t i g e n SP-10. B i o l . Reprod. 42, 693 Yanagimachi, R., Yanagimachi, H. and Rogers, B.J. (1976) The use of zona-free animal ova as a t e s t system f o r the assessment of the f e r t i l i z i n g c a p a c i t y o f human spermatozoa. B i o l . Reprod. 15, 471 Yanagimachi, R. (1981) Mechanisms of f e r t i l i z a t i o n i n mammals, i n : F e r t i l i z a t i o n and Embryonic Development In V i t r o . Plenum, New York. pp. 81 Yanagimachi, R. (1984) F e r t i l i z a t i o n i n mammals. Tokai J . Exp. C l i n . Med. 9, 81 Yang, Y-S, Rojas, F . J . and Stone, S.C. (1988) Acrosome r e a c t i o n of human spermatozoa i n zona-free hamster egg p e n e t r a t i o n t e s t . F e r t i l . S t e r i l . 50, 954. Young, R. A., and Davis, R. W. (1983) E f f i c i e n t i s o l a t i o n of genes by u s i n g antibody probes. Proc. N a t l . Acad. S c i . (USA) 80, 1194. 201 APPENDIX 1. The formula of s o l u t i o n s i n ELISA are summarized as f o l l o w s : (1) . B l o c k i n g b u f f e r T r i s - H C l (pH 8.0) 0.1 M BSA 0.25 % Sucrose 2 % Thimerosal 0.1 % (2) . S u b s t r a t e s o l u t i o n C i t r a t e - p h o s p h a t e (pH 5.0) 0.1 M 3,3',5,5' t e t r a m e t h y l b e n z i d i n e 0.03 % H 2 0 2 • 0.02 % 2. The formula o f stock s o l u t i o n s mentioned i n SDS-PAGE are summarized as f o l l o w s : (1) . S t a c k i n g g e l acrylaraide s o l u t i o n acrylamide 28.38 gm b i s a c r y l a m i d e 1.62 gm dH 20 t o 100 ml (2) . S t a c k i n g T r i s - H C l s o l u t i o n T r i s base 6.06 gm SDS 0.4 gm pH t o 6.8 wit h HCl dH 20 t o 100 ml (3) . Running g e l acrylamide s o l u t i o n acrylamide 3 0.0 gm b i s a c r y l a m i d e 0.8 gm dH 20 t o 100 ml (4) . Running T r i s - H C l s o l u t i o n T r i s base 18.17 gm SDS 0.40 gm pH t o 8.8 with HCl dH 20 t o 100 ml (5) .. E l e c t r o p h o r e s i s b u f f e r (10 X) ' T r i s base 30 gm g l y c i n e 144 gm SDS 10 gm (6) . Sample b u f f e r (2 X) • 1 M T r i s - H C l , pH 6.8 1.25 ml 10 % SDS 4 ml g l y c e r o l 1 ml bromophenol blue 20 mg B-mercaptoenthanol 0.4 ml B r i n g volume t o 10 ml with dH 20 202 3. The formula of s t o c k s o l u t i o n s mentioned i n 2D-gel e l e c t r o p h o r e s i s above are summarized as f o l l o w s : (1) . Ampholine mixture pH range 3.5-10:4-6:5-8 = 1:1:3 (2) . Gel mixture (5 ml) urea 2.5 gm 30 % acrylamide 0.7 ml ( f o r upper g e l of SDS-PAGE) g l y c e r o l 0.25 ml dH 20 1.25 ml ampholine mixture 0.25 ml 10 % ammonium p e r s u l f a t e 10 u l TEMED 10 u l (3) . Sample b u f f e r urea 20 % NP-40 ampholine mixture 1 M DTT dH 20 t o 1 ml (4) . SDS sample b u f f e r 1 M T r i s - H C l pH 6.8 1.8 ml 10 % SDS 6.0 ml 2-mercaptoenthanol 1.5 ml 10 % g l y c e r o l 3.0 ml dHoO t o 30 ml 0.57 gm 0.1 ml 0.05 ml 0.025 ml 4. The formula of stock s o l u t i o n s mentioned i n dot b l o t assay are l i s t e d as f o l l o w s : (1) . TBS 50 mM T r i s - H C l (pH 7.9) 150 mM NaCl (2) . TBST TBS p l u s 0.05 % Tween-20 (3) . S u b s t r a t e s o l u t i o n 30 mg (W/V) urea peroxide i n 50 ml 0.1 M T r i s - H C l , pH 8.0 30 mg (W/V) 4 - C h l o r p - l - n a p t h o l i n 10 ml methanol mix w e l l b e f o r e use 5. The formula of stock s o l u t i o n s mentioned i n Western b l o t are summarized as f o l l o w s : ( 1 ) . B l o c k i n g s o l u t i o n 3 % BSA and 0.1 % TMS i n TBST s o l u t i o n 203 (2) . T r a n s f e r b u f f e r 25 mM T r i s base 192 mM g l y c i n e A f t e r T r i s and g l y c i n e are d i s s o l v e d , add 20 % (V/V) methanol pH t o 8.3 wit h HCl (3) . Amido b l a c k s t a i n i n g and d e s t a i n i n g s o l u t i o n a. s t a i n i n g s o l u t i o n : 0.1 % (W/V) amido b l a c k i n 45 % methanol, 45 % H 20 and 10 % a c e t i c a c i d b. d e s t a i n i n g s o l u t i o n : s t a i n i n g s o l u t i o n without amido b l a c k 6. The formula o f stock s o l u t i o n s mentioned i n mouse i n v i t r o f e r t i l i z a t i o n are l i s t e d as f o l l o w s : (1) . BWW medium (Bigger e t a l . , 1971) NaCl 0.554 gm KC1 0.0356 gm C a C l 2 0.0189 gm KH 2P0 4 0.0162 gm MgS0 4 7H 20 0.0294 gm Dextrose 0.1 gm p y r u v i c a c i d 0.003 gm (Na s a l t ) NaHC0 3 0.2106 gm BSA 0.3 5 gm D L - l a c t i c a c i d 0.37 ml 1 M Hepes, pH 7.4 2 ml 0.5 % phenol r e d 0.05 ml Pen-Strep 1 ml ddH 20 t o 100 ml F i l t r a t e the medium by u s i n g 0.22 urn f i l t e r (2) . IVF s t a i n i n g s o l u t i o n 0.25 % Lacmoid i n 45 % a c e t i c a c i d (3) . IVF d e s t a i n i n g s o l u t i o n 0.25 % g l y c e r o l i n 20 % a c e t i c a c i d 7. The s t o c k s o l u t i o n r e q u i r e d f o r t i s s u e DNA e x t r a c t i o n are l i s t e d as f o l l o w s : (1) . TE b u f f e r T r i s - H C l pH 8.0 10 mM EDTA 1 mM (2) . TNE b u f f e r TE b u f f e r p l u s 0.1 M NaCl 204 ( 3 ) . "Phenol" means phenol e q u i l i b r a t e d with b u f f e r and c o n t a i n i n g 0.1 % hy d r o x y q u i n o l i n e and 0.2 % 2-mercaptoethanol. (4 ) . "Chloroform" means a 24:1 (v/v) mixture of c h l o r o f o r m and isoamyl a l c o h o l . 8. The st o c k s o l u t i o n used f o r bacteriophage DNA e x t r a c t i o n are l i s t e d as f o l l o w s : (1) . LB medium Bacto-tryptone' 10 gm Bacto-yeast e x t r a c t 5 gm NaCl 10 gm dH 20 t o 1 l i t e r A d j u s t pH t o 7.5 with NaOH (2) . SM b u f f e r NaCl 5.8 gm MgS0 4 7 H 90 2 gm 1 M T r i s - H C l (pH 7.5) 50 ml 2 % g e l a t i n 5 ml (3) . TE b u f f e r 10 mM T r i s - H C l (pH 7.5) 1 mM EDTA (pH 8.0) A l l the s o l u t i o n s were s t e r i l i z e d by a u t o c l a v i n g p r i o r t o use. 9. The stock s o l u t i o n used i n e x t r a c t i o n of plasmid DNA are l i s t e d as f o l l o w s : (1) . S o l u t i o n I 50 mM glucose 25 mM T r i s - H C l (pH 8.0) 10 mM EDTA (2) . S o l u t i o n I I 0.2 M NaOH 1 % SDS (3) . S o l u t i o n I I I 5 M potassium a c e t a t e (pH 4.8) 60 ml A c e t i c a c i d 11.5 ml dH 20 28.5 ml 10. The compositions of s o l u t i o n s used f o r RNA e x t r a c t i o n are l i s t e d as f o l l o w s : ( 1 ) . S o l u tion.D Guanidinium t h i o c y a n a t e 4 M 205 Sodium c i t r a t e , pH 7 25 mM S a r c o s y l 0.5 % 2-mercaptoethanol 0.1 M 11. The formula of s o l u t i o n s mentioned i n n i c k t r a n s l a t i o n are summarized as f o l l o w s : (1) . dNTP mixture 500 uM dATP 2 u l 500 uM dGTP 2 u l 500 uM dTTP 2 u l (2) . 10 X n i c k t r a n s l a t i o n b u f f e r 0.5 M T r i s - H C l pH 7.2 0.1 M MgS0 4 1 mM DTT 500 ug/ml BSA {3). STE b u f f e r 10 mM T r i s - H C l pH 8.0 0.1 M NaCl 1 mM EDTA 12. The st o c k s o l u t i o n s used i n Southern b l o t h y b r i d i z a t i o n are l i s t e d as f o l l o w s : (1) . 20 X SSC NaCl 175.3 gm Sodium c i t r a t e 88.2 gm dH 20 t o 1000 ml a d j u s t pH t o 7.0 (2) . H y b r i d i z a t i o n b u f f e r Na 2HP0 4 pH 7.2 0.25 M SDS 7.0 % dextran s u l f a t e 7.5 % 13. The s o l u t i o n s used i n immunoscreening are l i s t e d as f o l l o w s : (1) . A v i d i n - b i o t i n y l a t e d h o r s e r a d i s h peroxidase complex a v i d i n s o l u t i o n 40 u l b i o t i n y l a t e d h o r s e r a d i s h peroxidase 40 u l i n 10 ml TBST b u f f e r T h i s mixture i s incubated a t room temperature f o r 30 minutes before use. (2) . Peroxidase s u b s t r a t e s o l u t i o n 4 - c h l o r o - l - n a p t h o l (3 mg/ml) i n methanol 2 ml TBS p l u s 0.01 M i m i d a z o l e 10 ml 30 % hydrogen peroxide 5 u l 206 

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