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Evaluation of anti-sperm monoclonal antibodies as biomarkers to assess bull sperm capacitation, acrosome… Ambrose, Divakar Justus 1996

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EVALUATION OF ANTI-SPERM MONOCLONAL ANTIBODIES AS BIOMARKERS TO ASSESS BULL SPERM CAPACITATION, ACROSOME REACTION AND FERTILITY IN VITRO by DIVAKAR JUSTUS AMBROSE B.V.Sc, Tamil Nadu Agricultural University, Coimbatore, India, 1982 M.V.Sc, Tamil Nadu Agricultural University, Coimbatore, India, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1995 ® Divakar Justus Ambrose, 1995 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 r^ KU no<sX The University of British Columbia Vancouver, Canada Date K/gyeryiW 5" DE-6 (2/88) ABSTRACT Anti-sperm monoclonal antibodies (mAbs) were evaluated as biomarkers to assess capacitation, acrosome reaction, cryodamage and fertility of bull spermatozoa in vitro. Three anti-human sperm mAbs crossreacting with bull sperm antigens were identified. They showed time-dependent changes in binding to bull spermatozoa incubated under capacitation conditions when assessed by indirect immunofluorescence, with maximum binding at 4 h. Bull and human sperm antigens recognized by one mAb (HS-11) were isolated and their immunological relatedness shown by ELISA. The possible relationship between fertility and the binding of HS-11 to bull (n=8) spermatozoa was tested in a bovine in vitro fertilization system. In vitro fertility based on cleavage of oocytes and mAb-binding to spermatozoa at 4 h incubation under capacitation conditions were correlated (r=0.43; n=32; P<0.05), but in vivo fertility and sperm-HS-11 binding were not correlated. In the next set of experiments, 15 mAbs specific to bull sperm antigens were generated. The mAbs were sperm-specific, but not species-specific. The mAbs identified five distinct antigenic domains of bull spermatozoa. Seven of the 13 mAbs tested, recognized bull sperm proteins of >200 kDa in western blots. Three detected more than one protein band (40-200 kDa), while three recognized none. Thirteen of the 15 mAbs were tested for their influence on bovine sperm-zona interactions in vitro. Sperm-zona binding was not affected by 12 mAbs. However, sperm-zona binding in the presence of one surface-reacting mAb was higher than the control (23.6+5.6 vs 10.0+2.4 sperm/zona, mean+SE; P<0.001). Four mAbs specific to intra-acrosomal antigens exhibited a time dependent increase (P<0.05) in binding to bull spermatozoa incubated under capacitation conditions. In contrast, the binding of mAbs specific to surface antigens decreased i i (P < 0.05) after 4 h incubation in the presence of heparin. Following induced acrosome reaction, a significant decrease (P<0.01) in the binding of acrosome-specific mAbs was observed. Four selected mAbs were then evaluated as possible indicators of cryodamage in frozen-thawed bull spermatozoa. Even though the mAbs showed higher binding to frozen-thawed spermatozoa, there was no conclusive evidence to support the hypothesis that the mAbs will be useful to detect live membrane-damaged spermatozoa. i i i TABLE OF CONTENTS A B S T R A C T i i T A B L E OF CONTENTS iv LIST OF FIGURES vi LIST OF PLATES vii i LIST OF TABLES ix ABBREVIATIONS x A C K N O W L E D G E M E N T S xi i DEDICATION xii i FOREWORD xiv C H A P T E R 1. G E N E R A L INTRODUCTION 1 C H A P T E R 2. A C O N C E P T U A L OVERVIEW 4 2.1. STRUCTURE OF T H E S P E R M A T O Z O O N 4 2.2. S P E R M C A P A C I T A T I O N 17 2.3. A C R O S O M E R E A C T I O N 20 2.4. FERTILIZATION 24 2.5. L A B O R A T O R Y PROCEDURES TO E V A L U A T E FERTILITY 25 2.6. TECHNIQUES FOR ASSESSING S P E R M S U R F A C E C H A N G E S 32 2.7. M O N O C L O N A L ANTIBODIES 36 C H A P T E R 3. IDENTIFICATION OF A N T I - H U M A N S P E R M M O N O C L O N A L ANTIBODIES CROSSREACTIVE TO B U L L S P E R M ANTIGENS 40 3.1. A B S T R A C T 40 3.2. INTRODUCTION . . . 41 3.3. M A T E R I A L S A N D METHODS 43 3.4. RESULTS 49 3.5. DISCUSSION 59 3.6. CONCLUSION 63 C H A P T E R 4. E V A L U A T I O N OF T H E A N T I - H U M A N S P E R M M O N O C L O N A L ANTIBODY HS-11 AS A M A R K E R TO ASSESS FERTILITY 64 4.1. A B S T R A C T 64 4.2. INTRODUCTION 65 4.3. M A T E R I A L S A N D METHODS 67 4.4. RESULTS 73 iv 4.5. DISCUSSION 78 4.6. C O N C L U S I O N 82 C H A P T E R 5. PRODUCTION A N D C H A R A C T E R I Z A T I O N OF M O N O C L O N A L ANTIBODIES TO B U L L SPERMATOZOA 84 5.1. A B S T R A C T 84 5.2. INTRODUCTION 85 5.3. M A T E R I A L S A N D METHODS 86 5.4. RESULTS 94 5.5. DISCUSSION 109 5.6. C O N C L U S I O N 114 C H A P T E R 6. F U N C T I O N A L C H A R A C T E R I Z A T I O N A N D E V A L U A T I O N OF A N T I - B U L L S P E R M M O N O C L O N A L ANTIBODIES FOR POTENTIAL BIOLOGICAL APPLICATIONS 115 6.1. A B S T R A C T 115 6.2. INTRODUCTION 117 6.3. M A T E R I A L S A N D METHODS 119 6.4. RESULTS 127 6.5. DISCUSSION 141 6.6. C O N C L U S I O N 145 C H A P T E R 7. F I N A L DISCUSSION A N D F U T U R E CONSIDERATIONS 146 7.1. S U M M A R Y OF FINDINGS 146 7.2. O V E R A L L DISCUSSION 149 7.3. F U T U R E CONSIDERATIONS 153 REFERENCES 156 A P P E N D I X A . COMPOSITION OF MODIFIED T Y R O D E ' S M E D I A , SP-TALP A N D F E R T - T A L P 176 A P P E N D I X B. STEPS I N V O L V E D I N BOVINE I N VITRO FERTILIZATION . . . 177 A P P E N D I X C. D U A L STAINING TECHNIQUE FOR VIABILITY A N D A C R O S O M A L STATUS ASSESSMENT 178 A P P E N D I X D. C E L L FUSION PROTOCOL 179 A P P E N D I X E . PROTOCOL FOR ASSESSING S P E R M A N T I G E N L O C A L I Z A T I O N B Y SCANNING E L E C T R O N MICROSCOPY 181 APPENDIX F. PROTOCOL FOR SPERM-ZONA BINDING A S S A Y 182 v' LIST OF FIGURES Figure 2.1. A schematic illustration of a mammalian spermatozoon 5 Figure 2.2. Schematic representation of the chief antigenic domains of mammalian spermatozoa 16 Figure 3.1. Percent binding (mean±SE) of the mAbs HS-9, HS-11 and HS-63 to fresh live bull spermatozoa, as determined by UFA, when they were co-incubated for 30-min periods at 0, 2, 4, 6 and 8 h of culture in H-TALP medium 52 Figure 3.2. Mean (+ SE) percent binding of the three monoclonal antibodies and that of PSA to bull spermatozoa, before and after LC treatment 53 Figure 3.3. The similarity in percent change of the binding of the mAbs HS-9, HS-11 and HS-63 between one another is illustrated 54 Figure 3.4. The similarity in the relationship between HS-11 and PSA in their ability to detect acrosome changes in human spermatozoa 56 Figure 3.5. Results of ELISA revealing the binding specificity between the mAb HS-11 and the isolated human sperm antigens HSA-11 and HSA-63 and the bull sperm antigen BSA-11 coated on microwells 57 Figure 4.1. The time-dependent changes in mAb HS-11 binding to frozen-thawed sperm of eight different bulls 75 Figure 4.2. The percent increase in acrosome reaction of sperm from 0 h to 4 h following LC treatment. Note that spermatozoa of bulls 158 and 230 showed the lowest increase in acrosome reaction 76 Figure 4.3. The linear relationship between HS-11 binding to spermatozoa and the cleavage rate of bovine oocytes fertilized in vitro after excluding bull 196 77 Figure 5.1. Binding difference of mAbs to spermatozoa of testicular and epididymal origin 103 Figure 5.2. The general binding trend of the mAbs of I BS-series to live uncapacitated (Live), induced acrosome-reacted (LC-AR) and methanol-fixed (Fixed) spermatozoa 105 Figure 6.1. Comparison of mean binding of spermatozoa to oocytes (number bound per zona) after incubation with IIBS-2, all other mAbs (mean), normal mouse serum (negative control) and immune mouse serum (positive control) 130 vi Figure 6.2. Time-dependent changes in the binding of 8 mAbs (I BS series) to bull spermatozoa, tested live, under capacitation conditions 131 Figure 6.3. Binding of the surface reacting mAbs to live bull spermatozoa incubated under capacitation conditions. At 4.5 h, the binding of IIBS-2 and IIBS-11 were significantly lower to the heparin treated samples in comparison with heparin-free samples 132 Figure 6.4. Acrosome status of control and LC treated bull spermatozoa at 0.5 h and at 4.5 h determined by mAbs, FITC-labelled PSA and Giemsa stain 133 Figure 6.5. Differences in the percent binding of mAb I BS-1 to fresh and frozen-thawed spermatozoa of five bulls (mean+SE) 137 Figure 6.6. Correlation between mAb-binding and percent dead acrosome-intact spermatozoa (r=0.86; P<0.01) 138 vi i LIST OF PLATES Plate 3.1. Time-dependent changes in the binding of the anti-human sperm mAb HS-11 to the acrosome region of live bull spermatozoa, demonstrated by UFA 51 Plate 3.2. Results of the SDS-PAGE of purified sperm antigens isolated by immunoaffinity chromatography from human and bull semen extract 55 Plate 4.1. Dual stained bull spermatozoa showing the four different states, namely, live acrosome-intact (LI), live acrosome-reacted (LR), dead acrosome-intact (DI) and dead acrosome-reacted (DR) 71 Plate 5.1. Hybridomas in semisolid medium 12 days after cell fusion 89 Plate 5.2. Five distinct antigenic domains of bull sperm identified by mAbs 96 Plate 5.3. Binding patterns of the 15 mAbs to bull sperm as determined by IIFA 97 Plate 5.4. Interspecies crossreactivity of selected mAbs as determined by IIFA 101 Plate 5.5. Binding of HS-11 to pig sperm acrosome and mid-piece shown by IIFA . . . 102 Plate 5.6. Differential binding of surface-reacting mAbs to live and methanol-fixed sperm 106 Plate 5.7. Scanning electron micrograph of a bull spermatozoon showing the localization sites of mAb II BS-2 107 Plate 5.8. Results of western blot analysis of bull sperm antigens probed with 13 different mAbs 108 Plate 6.1. Sperm-zona binding assay. Note Hoechst 33345 stained spermatozoa bound to the zona pellucida of bovine oocytes (x 400) 122 Plate 6.2. Binding of live spermatozoa to zona pellucida in sperm-zona binding assays (II BS-2 vs control) 129 Plate 6.3. Acrosomal status of bull spermatozoa detected by the mAb I BS-1 before and after LC treatment 134 Plate 6.4. Indiscriminate staining of bull spermatozoa by Hoechst 33342 at 0.1/ig/ml. . . 139 Plate 6.5. Differential staining of bull spermatozoa by Hoechst 33258 at 0.5 ^g/ml. . . . 140 vii i LIST OF TABLES Table 3.1. The influence of HS-11 and normal mouse IgG on cleavage of bovine oocytes 58 Table 4.1. A comparison of between-bull differences in HS-11 binding to spermatozoa at 4 h incubation in capacitation medium, and differences in cleavage rate of in vitro fertilized bovine oocytes 74 Table 5.1. Interspecies crossreactivity of the anti-bull sperm mAbs 100 Table 6.1. The influence of mAbs II BS-2 and II BS-11 on sperm-zona interaction . . . 127 Table 6.2. Binding of mAbs to fresh and frozen-thawed spermatozoa of different bulls 136 ix ABBREVIATIONS Al = artificial insemination ANOVA = analysis of variance ATP = adenosine triphosphate BS = anti-bull sperm monoclonal antibody (e.g. I BS-1, II BS-3) BSA = bovine serum albumin BSA-11 = bull sperm antigen (recognized by HS-11) CEIA = competitive enzyme immunoassay CTC = chlortetracycline DIC = differential interference contrast (microscopy) DMSO = dimethylsulfoxide DNA = deoxyribonucleic acid ECL = enhanced chemiluminescence ECS = estrus cow serum ELISA = enzyme linked immunosorbent assay FBS fetal bovine serum FITC = fluorescein isothyocyanate g = gravity g = gram h = hour(s) HAT = hypoxanthine aminopterin thymidine HRP = horse radish peroxidase HS = anti-human sperm monoclonal antibody (e.g. HS-9, HS-11) HSA = human sperm antigen (e.g. HSA-11 is the cognate antigen of mAb HS-11) IAM = inner acrosomal membrane IEM = immunoelectron microscopy Ig = immunoglobulin (IgA, IgG, IgM) IIF = indirect immunofluorescence IIFA = indirect immunofluorescence assay IMP = intramembranous particles IVF in vitro fertilization kDa = kilo Dalton LC = lysophosphatidylcholine mAb(s) = monoclonal antibody (or) monoclonal antibodies mg = milligram min = minute(s) ml millilitre mM millimolar MS = anti-mouse sperm monoclonal antibody (e.g. MS-4, MS-4) nm = nanometer OAM = outer acrosomal membrane PAGE — polyacrylamide gel electrophoresis PBS = phosphate buffered saline PEG = polyethyleneglycol PSA = pisum sativum agglutinin (a lectin) X PVDF = polyvinylidine difluoride SAS = statistical analysis system SDS = sodium dodecyl sulfate SE = standard error sec = second(s) SEIA = sandwich enzyme immunoassay SPM = sperm plasma membrane TALP = Tyrode's albumin lactate pyruvate 1* = microlitre /zm = micrometre fiM = micromolar = microgram xi ACKNOWLEDGEMENTS First of all, I wish to extend my appreciation and deep gratitude to Dr. R. Rajamahendran for consenting to be my thesis supervisor and for his keen interest, encouragement, guidance and support at every stage of the project. The successful production of monoclonal antibodies during this project could not have been accomplished without the cooperation and technical expertise of Dr. Gregory Lee. I thank him for serving on my advisory committee, for allowing unrestricted access to his laboratory facilities and for his help and guidance during the two cell fusion experiments. I am also thankful to Dr. Y.S. Moon and Dr. A.M. Perks for serving on my advisory committee and offering helpful suggestions. The technical assistance rendered by Mr. K. Sivakumaran and Ms. K. Selvalogini at various times during my training is gratefully acknowledged. I am grateful to Dr. T. Yoshiki, visiting fellow from the Shiga University of Medical Sciences, Japan, for his technical advice and assistance with cryosections. I also thank Ms. Lisa Johnson, my colleague, for her help in my last experiment. Several of my fellow graduate students, both past and present, have offered suggestions from time to time or assisted with statistical analysis of data. I sincerely appreciate their help and wish to extend my special thanks to Samuel Aggrey, John Baah, Patrick Charagu, Sami Ibrahim, Jamal Kurtu, Mohan Manikkam and Chris Taylor. This acknowledgement would be incomplete without thanking all the faculty and staff members of the Department of Animal Science. I am particularly grateful to Dr. D.M. Shackleton for his help in generating some of the schematic illustrations used in this thesis, to Dr. R.M. Beames for making time to read my thesis for editorial correctness and to Dr. K.M. Cheng for his assistance as the graduate coordinator. I appreciate Donna, Gilles, Luz, Maureen, Shenaz and Sylvia for their assistance with office/laboratory procedures. I must also thank the dairy barn crew for their assistance. Margaret Rasheed and Andrew Lee of the Andrology laboratory deserve special thanks for their friendly interactions, excellent sense of humour and helpful gestures. I am thankful to the BCAI Centre, Milner, for supplying the semen samples for this study. The helpful nature of the chief-technician Ms. Sonya Klemm, and other staff members of the semen processing laboratory at the BCAI centre is remembered and deeply appreciated. I am grateful to the University of British Columbia for awarding me the University Graduate Fellowship for the entire duration of my doctoral training, the Department of Animal Science for offering teaching assistantships, the Faculty of Graduate Studies for awarding a travel grant, and the Science Council of BC for a short-term research assistantship. At a personal level I am deeply indebted to Siva and Jamal. Perhaps things would have been very different without these two friends around. Thank you very much guys for all that you have done to me and my family to make our stay in Vancouver comfortable and memorable. I also wish to thank Dr. George Muthian and many other Indian, Canadian and International friends for their good wishes and assistance. The loving thoughts, encouragement and prayers of my parents, my in-laws, sister, brother and other relatives have been essential for the accomplishment of this task. I thank them all for their affection and the Lord Almighty for His abundant blessings. The completion of this dissertation would not have been possible without the love, understanding and support of my wife Chitra. She has been a wonderful source of strength and courage to me. I greatly admire her for voluntarily shouldering several of my domestic responsibilities during the past four years. I am very grateful to her and our children Prashant and Daphne for putting up with my long hours of absence from home. xii Dedicated to My Wife and Children In Deep Appreciation of Their Love and Understanding xiii FOREWORD This thesis is based on the following manuscripts, which have either been published or submitted for publication: 1. J.D. Ambrose, R. Rajamahendran, S. Kunanithy, C.Y.G. Lee (1994) Three anti-human sperm monoclonal antibodies recognizing a conserved acrosome antigen: markers to detect acrosome reaction in vitro. Mol Androl 6:281-294. 2. R. Rajamahendran, J.D. Ambrose, C.Y.G. Lee (1994) The anti-human sperm monoclonal antibody HS-11: a potential marker to detect bovine sperm capacitation and acrosome reaction in vitro. J Reprod Fertil 101:539-545. 3. J.D. Ambrose, R. Rajamahendran, K. Sivakumaran, C.Y.G. Lee (1995) Binding of the anti-human sperm monoclonal antibody HS-11 to bull sperm is correlated with fertility in vitro. Theriogenology 43:419-426. 4. J.D. Ambrose, R. Rajamahendran, T. Yoshiki, C.Y.G. Lee (1995) Anti-bull sperm monoclonal antibodies: I. Identification of major antigenic domains of bull sperm and manifestation of interspecies crossreactivity. J Androl (submitted). 5. J.D. Ambrose, R. Rajamahendran, C.Y.G. Lee (1995) Anti-bull sperm monoclonal antibodies: II. Influence on bovine sperm-zona interaction in vitro and binding changes to spermatozoa under capacitation conditions. J Androl (submitted). xiv CHAPTER 1 GENERAL INTRODUCTION Freshly ejaculated mammalian spermatozoa are incapable of fertilizing the eggs unless they have undergone a series of poorly understood physiological changes termed capacitation, followed by acrosome reaction, an exocytotic event. For capacitation to take place, spermatozoa must either reside in the female reproductive tract or be incubated in defined media under favourable conditions in vitro. The phenomenon of capacitation was first discovered independently by Austin (1951) and Chang (1951). Despite over four decades of research, carried out primarily in laboratory animal species, the mechanisms involved in the process of sperm capacitation and acrosome reaction have still not been clearly defined. Recent studies have shown that there is a relationship between fertility of bulls and the ability of their spermatozoa to undergo induced acrosome reaction in vitro (Parrish et al., 1985; Ax and Lenz, 1986; Whitfield and Parkinson, 1992). Even though the process of capacitation is still poorly understood, there is general agreement that only capacitated spermatozoa can undergo acrosome reaction (Parrish et al., 1988; Yanagimachi, 1988; Meizel et al., 1990). Methods to assess capacitation of bull spermatozoa in vitro, would therefore, be of significant advantage to the artificial insemination industry, possibly for the development of laboratory tests to predict fertility. The direct assessment of surface/sub-surface changes associated with capacitation is extremely difficult due to the complexity of the sperm membranes and the elusive nature of the membrane modifications. Consequently, little is known about capacitation related changes. One of the reasons for the dearth of such information, clearly, is the non-availability of specific l biomarkers to carry out meaningful investigations. In recent years, the use of anti-sperm monoclonal antibodies (mAbs) has led to interesting observations on the migration, modification or disappearance of sperm antigens in the guinea pig (Myles and Primakoff, 1984), mouse (Okabe et al., 1986; Fann and Lee, 1992), pig (Saxena et al., 1986b) and human (Wolf et al., 1983) during epididymal maturation in vivo or capacitation in vitro. Therefore, the use of mAbs as biomarkers to assess bull sperm capacitation and acrosome reaction in vitro may provide a basis for developing laboratory tests to predict bull fertility. Even though mAbs have previously been raised against bull spermatozoa (Chakraborty et al., 1985; Bowen, 1986) the usefulness of mAbs as biomarkers to assess bull sperm surface changes in-vito) and their possible relationship to fertility have never been investigated. The overall objective of this thesis project, therefore, is to identify and/or generate anti-sperm mAbs for assessing bull sperm surface changes associated with capacitation and acrosome reaction in vitro and to test for any possible relationship between sperm-mAb binding and fertility under in vitro conditions. The possible applications of mAbs to assess cryodamage in frozen-thawed bovine spermatozoa and their role in sperm-oocyte interaction will also be investigated. This thesis is presented in seven chapters. This brief introductory chapter is followed by a conceptual overview (chapter 2). Chapter 3 deals with the identification of mAbs that crossreact with bull spermatozoa, from a pre-existing panel of anti-mouse and anti-human sperm mAbs, the isolation of the cognate bull sperm antigen recognized by an anti-human sperm mAb, the establishment of their immunological relatedness by various immunoassays, and investigation into the usefulness of the mAbs in sperm function assays. It also includes investigations on the time-dependent changes in mAb-binding to live sperm incubated under capacitation conditions in vitro. Results of the investigations on variations in the binding of the anti-human sperm mAb 2 HS-11 to spermatozoa of different bulls and the relationship between HS-11 binding at 4 h and bull fertility are presented in chapter 4. Chapter 5 focuses on the production and characterization of anti-bull sperm mAbs specific to intra-acrosomal and surface antigens. Experiments focused on the evaluation of the anti-bull sperm mAbs as markers to assess sperm surface changes (capacitation, acrosome status, cryodamage) in vitro are presented in chapter 6. A summary of the findings, a general discussion and considerations for further research in this area using the mAbs identified during this project are included in the final chapter. 3 CHAPTER 2 A CONCEPTUAL OVERVIEW The purpose of this chapter is to briefly review the structure of the spermatozoon with particular reference to the organization of sperm membranes, the antigenic domains of sperm surface and their modifications during epididymal maturation, capacitation and acrosome reaction. In addition, the research methods currently available for assessing sperm surface changes, their relative merits and demerits and their importance with relevance to the bovine artificial insemination (Al) industry will be discussed. 2.1. Structure of the spermatozoon The mammalian spermatozoon is remarkably different from any other type of cell in its unique characteristics, form and function. The bull spermatozoon can be structurally divided into the head and tail (Saacke and Almquist, 1964a, 1964b; Garner and Hafez, 1987). It has an overall length of 68 to 74 /xm; the head being 8 to 10 /xm long, 4 to 5 /xm wide and 0.3 to 0.5 /xm thick (Sullivan, 1978). The anterior part of the tail is referred to as the mid-piece (about 10 /xm long and about 0.85 in diameter), while the posterior part of the tail is subdivided into the principal-piece (45 to 50 /xm long) and the end-piece (2 to 4 /xm long). The head and the tail are connected by a short (0.3 to 1.5 /tm) neck. 4 V c Plate 2.1. A schematic illustration of a mammalian spermatozoon. A) Mid-piece; B) Principal-piece; C) End-piece; 1) Plasma membrane; 2) Outer acrosomal membrane; 3) Acrosomal contents; 4) Inner acrosomal membrane; 5) Nuclear membrane; 6) Nucleus; 7) Centrioles; 8) Mitochondria; 9) Fibrous sheath; 10) Axoneme; 11) The "9+9+2" arrangement of microtubules in the axoneme. 5 2.1.1. Sperm organelles and membranes As in any other living cell, the outermost limiting structure of the spermatozoon is the plasma membrane. Underlying the plasma membrane is the acrosome surrounded by the outer and inner acrosomal membranes. The large nucleus carrying the genetic material of the sperm lies underneath the acrosome, enclosed by the nuclear membrane. The mitochondrial sheath wraps around the mid-piece region and is considered the "power house", supplying energy for the metabolic activities of the sperm. The structure of the sperm tail is similar to that described for flagella or cilia, with the classical 9+9+2 arrangement of axial fibre bundle (Saacke and Almquist, 1964b; Sullivan, 1978). A schematic illustration of a bull spermatozoon depicting the major structures and location of the membranes is presented in Figure 2.1. 2.1.1.1. The sperm plasma membrane (SPM) The cell membrane is a mosaic structure of fluid lipids and globular proteins playing a central role in cellular physiology, cell-to-cell communication, recognition and immunobiology (Douglas and Zuckerman, 1976). Sperm membranes, like those of other cells, are composed of a phospholipid bilayer containing other lipids and proteins required for membrane and cellular function (Foote and Parks, 1993). Freeze fracture studies on mammalian SPM have shown thepresence of intramembranous particles (IMP), which are capable of aggregation at specialized sites such as the gap junction, and of reversible aggregation under a variety of environmental conditions (Friend and Fawcett, 1974; Guraya, 1987; Suzuki, 1990). Interactions between the lipid, protein and carbohydrate components of the SPM and extracellular components are essential for sperm function. Phospholipids make up 70% of the total lipids in the boar SPM. Sterols are the next most abundant lipid with a cholesterol/phospholipid molar ratio of about 0.12 (Nikolopoulau et al., 6 1985). Glycolipids and diacyl glycerols are less abundant, with free fatty acid making up a small amount. The phospholipid/protein ratio is about 0.68 on a weight basis, suggesting that the amount of total protein and lipid in the SPM are about the same, though the amounts and types of lipids and the lipid/protein ratios may probably be different in various domains (Eddy, 1988). The amount of sterol in the anterior acrosomal region of the SPM is about four times more than that of the post acrosomal region (Bradley et al., 1980; Friend, 1982). Changes in the lipid content of SPM occur during maturation and capacitation and these may have substantial effects on the composition and function of the membrane in different domains (Eddy, 1988). 2.1.1.2. The Acrosome The acrosome is a large zymogen-secreting granule that sits as a cap over the nucleus in the anterior part of the sperm head (Figure 2.1). It also contains the hydrolytic enzymes typically found in primary lysosomes. The acrosome originates from the Golgi complex in the spermatid, and has two segments, the anterior acrosome (acrosomal cap) and equatorial segment (posterior acrosome). The portion of the anterior acrosome which extends beyond the anterior margin of the nucleus is referred to as the "apical segment", and the portion overlying the nucleus is called the "principal segment" (Fawcett, 1975). In spermatozoa of primates, bulls, boars and rabbits, the acrosome is relatively small with no appreciable extension beyond the nucleus, whereas in the guinea pig, the chinchilla and the ground squirrel, the acrosome carries a large apical segment (Fawcett, 1970; Eddy, 1988). Acrosin and hyaluronidase, which are major constituents of the acrosome, are enzymes unique to the mammalian sperm acrosome. Comprehensive lists of acrosomal enzymes have been presented by Guraya (1987) and Eddy and O'Brien (1994). Acrosin is a trypsin-like serine 7 proteinase. The enzyme is present in the acrosome as pro-acrosin, which is converted to the active form during acrosome reaction. Glycosaminoglycans are known to help in the conversion of pro-acrosin to acrosin. Though present mainly in the anterior acrosome (Garner and Easton, 1977) , acrosin may also be present in the inner acrosomal membrane (Green and Hockaday, 1978) . However, since acrosin is rapidly released following acrosome reaction, it has been suggested that the bulk of the acrosin may be in the soluble acrosomal matrix (Yanagimachi, 1988). Hyaluronidase present in the acrosome is distinct from the common lysosomal form, and like acrosin, appears to be a spermatogenic-cell-specific isozyme. This glycosidase is abundantiy present and predominantly located in the anterior acrosome in bull (Gould and Bernstein, 1975) and ram (Brown, 1981) spermatozoa. There is some evidence to show that some hyaluronidase may be bound to the inner acrosome membrane as well (Morton, 1975). Carbohydrate is a distinct component of the acrosomal matrix. Glycoproteins or carbohydrate-containing materials within the acrosomal matrix may aid in the conversion of inactive forms of acrosomal enzymes (e.g., proacrosin) to active forms (e.g., acrosin), before or during the acrosome reaction (Yanagimachi, 1988). 2.1.1.3. The Outer and Inner Acrosomal Membranes The inner acrosomal membrane (IAM) is closely applied to the anterior part of the nuclear envelope. The non-adherent outer acrosomal membrane (OAM) surrounds the remainder of the acrosome and underlies the SPM. The inner surface coat of the OAM has been isolated from bull spermatozoa (Olson et al., 1985) and shown to be composed of several proteins, including 3 high molecular weight glycoproteins (260 to 290 kDa). Olson et al. (1985) also found that lectins bind the inner 8 surface of the OAM and suggested that glycosylated molecules at the site may help to stabilize the membrane or play a functional role in membrane fusion events of acrosome reaction. A thin layer of glycoprotein covering the inner surface of the OAM may serve to hold vesiculated SPM and OAM together during the acrosome reaction (Huang and Yanagimachi, 1985). The development of the IAM begins during the early spermatid stage of differentiation. It tightly associates with the condensing sperm nucleus, and becomes exposed following the acrosome reaction (Huang and Yanagimachi, 1985). In early stages of acrosome formation, the IAM is in close apposition with the nuclear envelope. However, in later stages, substance accumulates through an unknown mechanism between these two membranes and is referred to as subacrosomal material (Franklin and Fussell, 1972). The IAM in mature spermatozoa is one of the membranes most resistant to chemical and physical disruption, including treatment with non-ionic detergents and sonication (Rahi et al., 1983; Huang and Yanagimachi, 1985). However, studies have shown that the boar sperm IAM is sensitive to proteinase treatment (Russell et al., 1980). Localization of glycoprotein molecules on the IAM of guinea pig sperm has been shown by lectin binding studies (Schwarz and Koehler, 1979). Freeze-fracture studies indicate that the IAM is very rich in dense IMPs, and it has been proposed that the crystalline array of these particles may represent zona lysin material (Koehler, 1975). The IAM does not directly "fuse" with the egg plasma membrane, but rather is engulfed into the egg in a phagocytic fashion and eventually disintegrates (Bedford, 1972). It has been suggested that requirements for rigidity during zona penetration have resulted in a loss in membrane fluidity or in the inability to form IMP-free areas, preventing the IAM from fusing directly with the egg plasma membrane (Huang and Yanagimachi, 1985). Two schools of thought prevail to explain mammalian sperm penetration: 1) enzymatic digestion and 2) mechanical forces (Yanagimachi, 1981). Green and Purves (1984), after biophysical considerations, suggested that the mechanical 9 force of the motile sperm in conjunction with the viscoelastic properties of the zona pellucida are sufficient to achieve zona penetration, even without the aid of enzymes. This, and the earlier finding that the acrosome-reacted hamster sperm penetrates the zona by "slicing through", leaving a thin sharply defined penetration slit (Austin and Bishop, 1958; Yanagimachi, 1966) strongly supports the latter school of thought and suggests that the rigid nature of the IAM may be crucial for successful sperm penetration. 2.1.1.4. The Nucleus and Nuclear Membranes The major components of the sperm head are the nucleus and acrosome. It also contains cytoskeletal structures and a small amount of cytoplasm. The volume of the haploid sperm nucleus is less than that of somatic cells, and its chromatin is highly condensed (Eddy, 1988). The sperm nucleus is enclosed by a nuclear envelope. The nuclear envelope is unusual in that nuclear pores are absent over most of the nucleus, and the two membranes of the nuclear envelope lie only 7-10 nm apart (Eddy, 1988). However, caudal to the posterior ring, the nuclear pores are abundant, and the two membranes are 40-60 nm apart as in most other cells (Fawcett, 1975; Eddy, 1988). A protein meshwork lining the inner surface of the nuclear envelope, referred to as the nuclear lamina, is thought to form the skeletal framework of the nuclear envelope and to serve as an anchoring site for chromatin. The nuclear lamina contains three closely related proteins lamins A, B and C, ranging in molecular weight from 60 to 70 kDa. Even though biochemical evidence for the presence of lamins in the mammalian sperm nucleus is lacking, it appears likely that lamins or related proteins contribute to the structure of the sperm nucleus (Eddy, 1988). 10 2.1.1.5. Cytoskeleton of sperm head The cytoskeletal components are located in two regions of the sperm head: a) sub-acrosomal and b) post-acrosomal. The sub-acrosomal and post-acrosomal cytoskeletons together are referred to as the "perinuclear theca". The perinuclear theca tend to retain the shape of the sperm nucleus when isolated, even in the absence of DNA and the acrosome. Therefore, it appears that the perinuclear theca may be responsible for determining the nuclear shape (Eddy, 1988). The sub-acrosomal cytoskeleton first appears in early spermatids and elongates during spermiogenesis. It is well defined and extensive in sperm with falciform heads (e.g., rodents), but is a minor component in other types of sperm. The formation of the post-acrosomal cytoskeleton begins in late spermiogenesis. Cytoskeletal components of the sperm head appear to have mechanical and functional roles in fertilization (Eddy, 1988). 2.1.1.6. The flagellum or tail The tail has three distinct segments, the middle piece, the principal piece and the end piece (Figure 2.1). The main structural features of the mammalian sperm tail are the axoneme, the mitochondrial sheath, the outer dense fibres and the fibrous sheath. The axoneme of the mammalian sperm tail extends the full length of the flagellum. It consists of two central microtubules surrounded by 9 microtubule doublets. The microtubules are composed of a-tubulin and /3-tubulin, closely related proteins of about 55 kDa. Dynein is another chief protein present in the microtubules (or their arms) with ATPase activity, which is believed to be responsible for the sliding forces generated between adjacent doublets of microtubules during flagellar movement. For more detailed information including the ultrastructure of the tail, please refer Saacke and Almquist (1964b) and Eddy and O'brien (1994). 11 2.1.2. Differences in testicular, epididymal and ejaculated spermatozoa When spermatozoa leave the testis, they are immotile and cannot fertilize an egg. Their fertilizing ability is acquired only during epididymal transport (Eddy et al., 1985; Yanagimachi, 1988; Lacham and Trounson, 1991; Fournier-Delpech and Thibault, 1993). The epididymis is about 40 m long in the bull and its main functions are transport, storage and functional maturation of spermatozoa. The total duration of sperm transport through the epididymis varies with species. In the bull, it takes about 14 days, about 3 to 5 days in the mouse and 11 days in the rat (Fournier-Delpech and Thibault, 1993). During this period of passage through the epididymis, spermatozoa undergo structural changes, alterations in cell surface components and also acquire motility. Androgen support to the epididymis is absolutely essential for normal epididymal sperm maturation (Garner and Hafez, 1987). Among structural changes, the migration and eventual loss of the cytoplasmic droplet and acrosome remodelling (in some species, e.g. guinea-pig, chinchilla and the pig-tailed macaque) are considered significant (Fawcett and Phillips, 1969). Other changes like the increase in the space between SPM and OAM (Bellve and O'brien, 1983), and modifications in the pattern and arrangement of IMPs (Suzuki, 1990), have been demonstrated. It has been suggested that such changes may have a possible role in promoting fusion between the two membranes later during acrosome reaction. The sperm nucleus develops a resistance to decondensation, and the acrosome becomes more tightly applied to the nucleus (Fournier-Delpech and Thibault, 1993) in the epididymis. Several alterations are known to occur in the surface components of spermatozoa during epididymal transport. Even though the total lipid content of the sperm cell decreases during epididymal maturation, the molar ratio of cholesterol to phospholipid increases in the periacrosomal region of the SPM (Bellve and Obrien, 1983; Parks and Hammerstedt, 1985). 12 There is, in fact, an increase in the unsaturated fatty acid content in the phospholipid fraction of the SPM. The increase in unsaturated fatty acid content and the decrease in total phospholipid content may be the reason for the increased sensitivity of epididymal and ejaculated spermatozoa to cold-shock, when compared to testicular spermatozoa (Bellve and O'brien, 1983). Studies have indicated that both addition and removal of surface proteins take place during epididymal transit (for reviews, see Bedford, 1975; Bedford and Cooper, 1978; Eddy, 1988; Yanagimachi, 1988). In the ram, some proteins of testicular origin become undetectable, whereas in the rat, ram, bull and chimpanzee there is an addition of new proteins to the sperm head (Yanagimachi, 1988; Fournier-Delpech and Thibault, 1993). Such binding of glycoproteins to the sperm surface during epididymal transport could account for changes in surface properties such as charge density and adhesiveness (Bellve and O'brien, 1983). Major differences seen in the topographic distribution of lectin binding sites between caput and cauda epididymal spermatozoa have also been reported (Friess and Sinowatz, 1984). The spermatozoa remain motionless in the caput epididymis, with a minimum vibratory flagellar movement. However, they attain the ability to move linearly on reaching the corpus epididymis (Fournier-Delpech and Thibault, 1993). Proteins secreted in the epididymis that are responsible for the initiation of progressive motility in spermatozoa are called forward-motility-proteins. The bovine forward motility protein is a heat-stable glycoprotein with a molecular weight of 37.5 kDa (Bellve and Obrien, 1983). Further, the spermatozoa become capable of binding to the zona pellucida of the oocyte only after they have passed through the epididymis (Garner and Hafez, 1987). Even though glycoproteins that mediate species-specific sperm-zona binding are intrinsic components of the plasma membrane, for these glycoproteins to become functional, apparently, there is a need for additional components of epididymal origin to be incorporated into the SPM. 13 In general, spermatozoa removed from the cauda epididymis would fertilize eggs in vitro much more readily than ejaculated spermatozoa. However, epididymal spermatozoa become "infertile" once they are exposed to seminal plasma. Many proteins of the accessory glands secreted into the seminal plasma bind to spermatozoa and prevent them from undergoing capacitation and acrosome reaction (Miller et al., 1990; Nass et al., 1990; Manjunath et al., 1993a). These surface factors described as "decapacitation factors" (Chang, 1957) must be either removed or masked for a spermatozoon to prepare itself for fertilization (Hunter and Nornes, 1969; Eddy, 1988; Yanagimachi, 1988). Such removal occurs during sperm transport in the female reproductive tract or can also be induced under in vitro conditions in a defined medium. From these observations it is clear that considerable differences exist between testicular, epididymal and ejaculated spermatozoa. Testicular spermatozoa, though fully formed at the time of release from the seminiferous tubules, undergo substantial modifications both in form and function during epididymal transport. Changes in the lipid content of the plasma membrane is notable. Some proteins are removed from the sperm surface, while several others are added. As a result of the various changes, the spermatozoa attain progressive motility and gain the ability to bind to zona pellucida, allowing the sperm to travel from the site of deposition (cervix or vagina) to the site of fertilization (oviduct), and to seek and eventually fertilize the egg. 2.1.3. Antigenic domains of the sperm plasma membrane The SPM is subdivided into regional domains that differ in composition and function. Thus the SPM is a mosaic of restricted domains that reflect the specialized functions of surface and cytoplasmic components of the spermatozoon. The domains are dynamic features that undergo changes in organization and composition during the life of the cell (Eddy, 1988). 14 Most surface domains are established during spermiogenesis while the round spermatid is being remodelled into the spermatozoon (Eddy, 1988). New surface antigens also appear in specific domains during maturation either through modification or unmasking of pre-existing molecules or by attachment of new molecules to acceptor sites already segregated into domains. The factors influencing domain formation in the mammalian SPM have been reviewed by Eddy and O'brien (1994). The major domains of the head region in most mammalian spermatozoa are the anterior acrosome (acrosomal cap), equatorial segment (posterior acrosome), and the post acrosomal region (Figure 2.2). In addition to the above major domains, there are the less well defined anterior band (situated between the anterior acrosome and the equatorial segment), serrated band (girdles the sperm head at the posterior margin of the equatorial segment) and posterior ring (lies at the junction between head and tail), the latter apparently forming a tight seal between the cytoplasmic compartments of the two main parts of the sperm. Further, the plasma membrane overlying the flagellum is separated into domains overlying the middle piece and the posterior tail by the annulus, which is a fibrous ring that surrounds the components of the axoneme and is firmly attached to the membrane. Several studies have identified the antigenic domains of spermatozoa of different species using monoclonal antibodies (mAbs) in recent years. Of these, the reports by Primakoff and Myles (1983) and Saxena et al. (1986b) could be considered important, with more than 50 mAbs being used in each study to map the surface of guinea pig and boar spermatozoa respectively. Five antigenic domains were identified in guinea pig spermatozoa, while up to 16 domains were identified in boar spermatozoa. Saccharide molecules are distributed with a regional heterogeneity on the sperm surface, as indicated by lectin-binding studies (Edelman and Millette, 1975; Ahluwalia et al., 1990). 15 A anterior acrosome A -equatorial segment V post-acrosomal region V • J, anterior band serrated band posterior ring annulus Figure 2.2. Schematic representation of the chief antigenic domains of mammalian spermatozoa. 16 Large numbers of lectin-binding sites have been identified in spermatozoa (2.5xl06 to 4.9xl07 sites per cell), occurring with higher density on the head, than on the tail (Edelman and Millette, 1975). Several mechanisms have been suggested in the restriction of domain movements within the SPM from one region to another. Some of the possible ways proposed are: a) the existence of a membrane barrier to surface component movement at the domain boundary, b) thermodynamic partitioning of molecules into a specific region, and c) mobility restriction occurring through interactions of intramembranous components with molecules outside or inside the membrane. It has been suggested that some surface domains may even be associated with underlying cell organelles (Eddy, 1988). 2.2. Sperm capacitation As discussed in section 2.1.2., testicular sperm which are non-motile and non-fertile when released from the seminiferous tubules, attain the ability for progressive motility, zona-binding and fertilization during epididymal transport. However, the fertilizing ability of spermatozoa is temporarily lost at ejaculation due to the addition of "decapacitation factors" from the seminal plasma. Spermatozoa, therefore, must reside in the female reproductive tract (in vivo) or in a defined medium (in vitro) for a certain period of time, and undergo a series of physiological changes, including the removal of the "decapacitation factors", in order to "regain" their fertilizing ability. These changes which render the ejaculated spermatozoa capable of fertilization are collectively called "capacitation". Capacitation is a reversible phenomenon, as capacitated spermatozoa can be decapacitated by re-introducing them to seminal plasma components. 17 2.2.1. Capacitation related changes in spermatozoa Several sperm surface modifications are known to occur during capacitation. Components of the seminal plasma collectively referred to as "decapacitation factors" prevent capacitation and must be removed, modified or masked to facilitate capacitation and acrosome reaction (Yanagimachi, 1988; Manjunath et al., 1993a). It has been shown that an influx of extracellular Ca 2 + is absolutely essential for acrosome reaction to happen both in vertebrates (Yanagimachi and Usui, 1974; Singh et al., 1978; Fraser, 1987) and invertebrates (Kazazoglou et al., 1985). Calmodulin is an acidic, low molecular weight protein known to regulate many cellular processes in a calcium-dependent manner in eukaryotic cells (Means et al., 1982). In bovine spermatozoa, calmodulin is bound to the inner surface of the SPM (Manjunath et al., 1993b) and participates in the regulation of events involving Ca 2 + transport during capacitation and the acrosome reaction. Heparin (in vitro) and heparin-like substances of the female reproductive tract (in vivo) are known to influence the calcium regulatory role of calmodulin through their interactions with bovine seminal plasma proteins. Recent studies including 125I-labelled calmodulin gel overlay procedures suggest that during capacitation Ca 2 + uptake could follow the decreased binding of calmodulin to specific sperm proteins (Leclerc et al., 1989; Leclerc et al., 1990; Manjunath et al., 1993b). Loss of cholesterol from SPM is another important event during capacitation (Davis, 1981; Langlais and Roberts, 1985; Ehrenwald et al., 1988a; 1988b), leading to increased membrane permeability (Sidhu and Guraya, 1989). Studies have confirmed the importance of cholesterol acceptor molecules (such as BSA or blood serum) in the capacitation medium for successful capacitation to take place (see Sidhu and Guraya, 1989 for references). Albumin, a multiple ligand carrier, has been shown to mediate sperm cholesterol efflux during in vitro capacitation in many species including the bovine (Ehrenwald et al., 1988a; Langlais et al., 18 1988; Parrish and First, 1991). Similarly, very low-, low-, and/or high-density lipoproteins of serum and follicular fluid have been identified as sterol acceptor molecules (Langlais et al., 1988). In the absence of a sterol acceptor, or in the presence of an agent such as glucose that would prevent sterol acceptance, capacitation cannot occur (First and Parrish, 1987; Florman and First, 1988; Parrish et al., 1989). During capacitation, IMPs migrate to form areas with and without IMPs. Fusion between the SPM and the OAM will begin in particle-free areas. These changes are mostly limited to the SPM overlying the acrosomal region. An increase in the intracellular pH is also known to occur during bovine sperm capacitation (Vredenburgh-Wilberg and Parrish, 1995). Another significant change that has been observed with most mammalian spermatozoa is an altered motility pattern, termed hyperactivation. Hyperactivation involves conspicuous and vigorous motility, with increased flexion of the flagellum, increased amplitude of flagellar waves and a reduced frequency of beat, resulting in a "whiplash" type of sperm movement (Yanagimachi, 1981). Recent studies indicate that even though capacitation-related changes in motility patterns do occur in bull spermatozoa, they display a different form of hyperactivity than what is typically seen in the laboratory animal species (Mcnutt, 1990). 2.2.2. Functional significance of sperm capacitation An important feature of capacitation in the female tract is believed to be a gradual removal or alteration of the protective glycoprotein coats (added to sperm surface during epididymal transport and at ejaculation), especially in the region of the acrosome. The removal or alteration of the coats would result in an exposure of sperm receptor sites, allowing spermatozoa to interact specifically with egg receptors and to undergo the acrosome reaction upon contact with the outer coats of the egg (Yanagimachi, 1990). The recent advances in in 19 vitro fertilization systems in several mammalian species, and the advancement of technologies that aid in the study of molecular events have considerably increased the understanding of the biochemical and molecular events associated with sperm capacitation. Despite this, a complete understanding of sperm capacitation is still lacking. However, there is consensus among researchers that even though capacitation involves no discernible morphological change, it is an essential pre-requirement for the occurrence of the acrosome reaction. Under in vitro conditions, the accomplishment of fertilization certainly means that spermatozoa underwent capacitation successfully. Unsuccessful in vitro fertilization, on the other hand, does not necessarily mean that the spermatozoa failed to undergo capacitation (Yanagimachi, 1988). Similarly, the acrosome reaction can also be taken as a reliable indicator of sperm capacitation, because spermatozoa do not undergo acrosome reaction unless they have been capacitated (Parrish et al., 1988; Yanagimachi, 1988). It is extremely important though, while making such conclusions, to remember that unusual conditions or special reagents may induce the acrosome reaction bypassing capacitation. 2.3. Acrosome reaction The acrosome reaction is a terminal (non-reversible) structural modification to the spermatozoon, and is essential for successful fertilization. Ultrastructural studies have shown that the acrosome reaction involves the fusion, vesiculation and loss of the OAM and its overlying SPM and the release of acrosomal matrix material (Meizel, 1984), resulting in the display of a new cell surface domain in the apical region of the sperm head (Florman and First, 1988). 20 2.3.1. Significance of acrosome reaction The eggs of most mammals are surrounded by a thick glycoprotein coat, commonly referred to as the zona pellucida, which the sperm must cross before reaching the egg plasma membrane. The zona pellucida is further surrounded by cumulus cells and their matrix. The acrosome reaction, therefore, has two major purposes: a) to help the sperm with the penetration of zona pellucida and b) to bring about fusion of the exposed sperm-surface with the egg plasma membrane. 2.3.2. Events associated with acrosome reaction As detailed in sections 2.1.2 and 2.2., the SPM contains numerous protein particles integrated in the membrane, each with its own specific function. Some of these proteins regulate ion transport. Many of them have a strong affinity for extracellular materials, and therefore absorb materials (e.g. glycoproteins) from within the testis during their passage through the epididymis and when exposed to seminal plasma at ejaculation. During capacitation, these absorbed materials are removed, enabling the protein particles in the membrane to move freely in the lipid bilayer (Yanagimachi, 1990). Maintaining a high intracellular concentration (140 mM) of K + , low concentration (5-10 + 2+ mM) of Na and a very low concentration (0.15 ^M) of Ca is important for preventing the spermatozoa from undergoing premature acrosome reaction. This is accomplished, at least partly, by the N a + / K + ATPase (pumping N a + out and K + into the cell) and Ca 2 + ATPase (pumping Ca 2 + out of the cell) systems. Many studies, including the most recent publication by Fraser et al. (1995) have shown that an increase in intracellular Ca 2 + is perhaps the most important pre-requisite for acrosome reaction to take place. Since acrosome reaction can be 21 induced forcibly in uncapacitated spermatozoa if Ca 2 + is driven into the cell by other means (Yanagimachi, 1975), it has been suggested that the primary role of capacitation could be to regulate the timing of Ca 2 + influx. Since the functional significance of the acrosome reaction is to ensure successful fertilization, it may not be reasonable to consider any site in the female reproductive tract other than the immediate vicinity of the egg for induction of acrosome reaction. But studies have shown that even locations such as the uterus (Wooding, 1975; Sidhu et al., 1986), far away from the site of fertilization, definitely play a role in the induction of acrosome reaction in bovine spermatozoa. Since spermatozoa cannot survive long after acrosome reaction, acrosome reactions occurring in distant locations such as the uterus may not really be of any functional significance to the ultimate event of fertilization; instead, it could be one of nature's mechanism of removing surplus spermatozoa from the competition for fertilization. Recent studies on the interactions between bovine spermatozoa and oviductal cells and follicular fluid have shown (Mcnutt, 1990; Ellington et al., 1991; Guyader and Chupin, 1991; King et al., 1994) that acrosome reactions could be induced by either specific factor(s) or by cell-cell interactions. Studies in the bovine as well as other species indicate that cumulus oophorus, zona pellucida and a host of molecules present in the female reproductive tract are involved in the induction of acrosome reaction (Meizel, 1985; Cross et al., 1988; Florman and First, 1988; Meizel et al., 1990; Carrell et al., 1993). Numerous other molecules (excluding components of female reproductive tract secretory products, cumulus oophorus and zona pellucida) have been reported to be involved in the regulation of acrosome reaction (see Yanagimachi, 1988). Evidence has also been presented to suggest that during sperm differentiation about 50% of mouse sperm are programmed to undergo premature acrosome reaction (Brown et al., 1989). It is clear from the evidence available that there is no one single 22 factor responsible for inducing acrosome reaction, but there could be several. There is, however, strong evidence to implicate heparin and heparin-like substances (glycosaminoglycans in general) of the female reproductive tract secretions as important factors in triggering the acrosome reaction in the bovine spermatozoon (Handrow et al., 1982; Lenz et al., 1982; Lenz et al., 1983; Lee et al., 1985; Miller and Ax, 1990). Excellent reviews have appeared on the kinetics of the mammalian sperm acrosome reaction (Meizel, 1984; Sidhu and Guraya, 1989; Yanagimachi, 1990; Brucker and Lipford, 1995). Based on the information presently available, the sequence of events that appear to culminate in acrosome reaction are as follows: Heparin or other glycosaminoglycans, or glycoproteins of the zona pellucida, bind to spermatozoa during capacitation and initiate Ca 2 + influx. Though it has not been clearly established how the Ca 2 + influx is initiated, it has been suggested (Yanagimachi, 1990) that the glycosaminoglycan-receptors on sperm surface could be Ca2+-carrier proteins, and upon activation the receptors may facilitate the diffusion of extracellular Ca 2 +. In addition to calmodulin, caltrin (a low molecular weight basic protein of seminal plasma origin) has also been implicated in the regulation of calcium transport in bovine spermatozoa (Lardy et al., 1986; Sitaram et al., 1986; San Agustin and Lardy, 1990; Clark et al., 1993). Fournier-Delpech and Thibault (1993) have suggested that heparin and heparin-like substances may remove caltrin from sperm surface, thus playing a direct role in Ca 2 + influx. A massive build-up of Ca 2 + turns off the Na +/K +ATPase pump, leading to an influx of Na + which in turn results in the efflux of H + , causing an increase in the intracellular pH. Upon entering the cell, Ca 2 + acts on the SPM (from inside) and OAM (from outside), leading to the activation of membrane bound phospholipase-A2 and phospholipase-C. The phospholipases in turn attack the phospholipids present in the membrane, leading to the release of fusogenic lysophospholipids, fatty acids and diacylglycerols. The fusogenic lysophospholipids bring about 23 fusion of the SPM and OAM, resulting in the vesiculation of membranes, allowing the Ca 2 + to enter the acrosomal matrix. As a result of Ca 2 + entry into the acrosome and subsequent H + loss, the acrosomal pro-acrosin is converted to enzymatically active acrosin, which disperses the acrosomal matrix containing various enzymes and lysins, resulting in the acrosome reaction. Once acrosome-reacted, spermatozoa must quickly penetrate the zona pellucida and fuse with the egg. If not, they will die very quickly, since gradual depletion of ATP and excessive accumulation of Ca 2 + within the cell are inevitable (Yanagimachi, 1990). 2.4. Fertilization Fertilization is the process whereby haploid male and female gametes, unite to form a diploid zygote, with the potential to produce a complete individual (Fraser and Ahuja, 1988; Longo, 1990). It has been shown that the mouse spermatozoa first loosely attach themselves to the zona pellucida. The O-linked oligosaccharide end of the zona protein ZP3 then interacts with a glycoprotein ligand of the spermatozoon, leading to its tight binding. The peptide portion of the ZP3 protein is then known to promote acrosome reaction, followed by sperm penetration of the zona, activation of the egg, and zona hardening. These and other events associated with fertilization, and the structural and functional aspects of the sperm receptors in the mouse have been described in detail (Florman and Wassarman, 1985; Wassarman, 1987, 1990, 1992; Bleil et al., 1988; Vazquez et al., 1989; Bleil and Wassarman, 1990; Mortillo and Wassarman, 1991). After the acrosome reaction, the sperm interacts with the external surface of the zona pellucida and eventually penetrates the zona by a combination of both mechanical and enzymatic forces (Yanagimachi, 1988). Once into the perivitelline space, the spermatozoon becomes associated with the plasma membrane of the egg and eventually fuses with it. The "cortical 24 reaction" (release of cortical granule material) takes place soon after, preventing the entry of any further spermatozoa. Decondensation of the sperm nucleus follows, which eventually forms a nuclear envelope to become the male pronucleus. Upon fusion with the spermatozoon, the egg "awakens" to initiate a series of changes that lead to differentiation and the formation of a new individual (Yanagimachi, 1988). Resumption of the second meiotic division, extrusion of the second polar body and the formation of the female pronucleus are major events that follow the fusion of the spermatozoon and egg. DNA synthesis in both the female and male pronuclei begins soon after the formation of the pronuclei. The fully developed male and female pronuclei then migrate towards the centre of the egg, where they finally meet. The nuclear envelopes disintegrate, allowing the chromosomes from both male and female pronuclei to mingle and the first mitotic division (cleavage) to occur. The fusion of the male and female pronuclei resulting in the mingling of chromosome material can be considered as the end of fertilization and the beginning of embryonic development. Parrish and First (1991) have suggested that bovine fertilization may be similar to that in the mouse. 2.5. Laboratory procedures to evaluate fertility Over the years, numerous attempts have been made to identify one or more semen characteristic(s) that can accurately predict the fertilizing potential of spermatozoa. The basic purpose of semen evaluation procedures is to ensure that only good quality semen is used for Al purposes. In other words, all semen evaluation procedures have the fundamental purpose of either predicting or enhancing the ability of the sperm to fertilize an oocyte. Even though the most valid test of sperm fertilizing ability is a viable pregnancy and a normal offspring following in vivo insemination (Bavister, 1990), the availability of simple in vitro tests that can foretell 25 sperm fertilizing ability, will be of great practical significance. A number of parameters would have to be considered for this purpose. The important ones are discussed here. 2.5.1. Motility, morphology and viability Among the parameters necessary for a basic analysis of semen, sperm concentration, motility, viability and morphology estimates would be considered the most important. The semen quality traits that are viability-related or of a morphological nature (see Saacke, 1982 for classification) could be assessed either by direct microscopic examination of semen samples or after simple staining procedures. Acceptable standards for a "probably fertile" specimen of bull semen are the presence of over 500 x 106 spermatozoa per ml and more than 50% of motile sperm making forward progression (Hafez, 1987). Several studies have compared the basic traits of semen quality with fertility estimates and correlations have ranged from zero to very high (Saacke, 1982). More recently, advanced technologies like computer-assisted semen analysis systems have become commercially available (Hamilton Thorn Research Inc., Danver, MA, USA; CellTrak, Motion Analysis Corporation, Santa Rosa, CA, USA; Cellsoft, CRYO-Resources Ltd., New York, NY, USA). Such systems allow an analysis of sperm translational movement, thus providing an alternative to subjective sperm motion analysis (Mcnutt, 1990). Budworth et al. (1988) used this technique to examine the relationship between sperm motility and fertility, and found a correlation between the two, and suggested that computerized motility analysis may be useful in the prediction of fertility of bull spermatozoa. In a more recent study, Bailey et al. (1994) found no correlation between any of the seven computer-determined motility parameters and in vivo fertility of cryopreserved bovine spermatozoa. Some of the advantages and disadvantages of the computer-assisted semen analysis systems have been discussed by Critser and Noiles (1993). 26 Other than motility, parameters like the live:dead ratios, acrosomal status and morphological abnormalities are considered important for predicting the fertilizing ability of bovine spermatozoa. Assessment of sperm viability have conventionally depended on supravital staining techniques. These are based on the principle that live cells possess intact plasma-acrosomal membranes through which these stains cannot pass, whereas dead cells will not have intact membranes and thus readily allow the passage of the macromolecular stain. Eosin-nigrosin, trypan blue or Congo red have been conventionally used for assessing membrane integrity of bull spermatozoa. Since the early reports on the usefulness of nucleic acid specific stains (bisbenzimide) for the sorting of live cells according to their DNA content (Arndt-Jovin and Jovin, 1977; Visser, 1980), commercially available stains such as Hoechst 33258 and Hoechst 33345 (Sigma, St. Louis, MO, USA) have been found useful for assessing membrane integrity of spermatozoa in a wide variety of species including human (Cross et al., 1986). Once acrosome-reacted, spermatozoa tend to die rapidly. In order to prolong the lifespan of spermatozoa, which may be essential for successful fertilization, the acrosomal integrity is very important. Saacke (1970) found a significant correlation between post-thaw motile life and maintenance of acrosome in bull spermatozoa. Direct counts of spermatozoa for intact acrosomes were found to be highly repeatable, with a coefficient of variance (CV) of 6% in comparison to a CV of 25% for motility estimates. The maximum permissible limits for abnormal spermatozoa in bull semen was set over 70 years ago. Williams and Savage (1925) found that if abnormal spermatozoa exceeded 18%, the fertility declined. Even though a wide variety of morphological abnormalities of spermatozoa have been reported, there is no clear experimental evidence of a relationship between specific morphological characteristics and fertility; however, a high frequency of abnormal spermatozoa has been associated with reduced fertility (Sullivan, 1978). The 27 maximum permissible limit for head abnormalities is set at 5 %, and for total abnormalities (all categories) it is 20%. Any sample exceeding these limits is considered unfit for AL 2.5.2. Biochemical parameters Measurements of metabolic activity of spermatozoa have also been considered as possible predictors of fertility. The use of metabolic tests such as oxygen uptake (Bishop and Salisbury, 1955), pyruvate oxidation (Melrose and Terner, 1953), fructolysis index (Secrist and Schultze, 1952), methylene blue reduction (Branton et al., 1951) and resazurin reduction (Erb and Ehlers, 1950) have been suggested, but were not found useful for routine evaluation of semen. The possibility of using enzyme-loss as a fertility-index was attempted by Pace and Graham (1970). They measured the release of glutamic-oxaloacetic-transaminase (GOT) from spermatozoa and found significant correlations between such measurements and fertility. The possible applications of assessing enzyme-loss in frozen-thawed spermatozoa and its relationship to fertility is under investigation (Buhr and Zhao, 1995; see section 2.5.3.). A kit for rapidly and conveniently assessing sperm viability by measuring ATP loss is now commercially available (Sperm Viability Test, FireZyme Diagnostic Technologies Limited, Halifax, NS, Canada). This test uses an enzyme, Luciferase (derived from fire-fly), which oxidizes luciferin (substrate) in proportion to the concentration of ATP present, resulting in the emission of light. Since ATP disappears within seconds following cell death, only the viable spermatozoa will contain ATP to contribute to the light-producing reaction. The light produced is measured in a bioluminometer. The kit priced around $ 3500.00 is currently being used on a trial basis by major Al companies. Published information on the usefulness of this test for making fertility estimates is still not available, but it is hoped that it may provide some reliable means of predicting fertility of semen samples. 28 2.5.3. Freezability and fertility It is well established that freezing and thawing procedures severely impair the cellular functions of spermatozoa (Parks and Graham, 1992), resulting in reduced fertility (Hammerstedt et al., 1990). Post-thaw sperm quality is important to the Al industry, as it directly relates to fertility and exhibits much variability between bulls (Elliot, 1978). A reliable index to predict fertility of young sires would therefore benefit the Al industry, considering the maintenance cost of a "probable-sires" during the 3-4 year period of progeny testing. Tests like cold shock index to assess the resistance of spermatozoa to low temperatures have been tried (Sullivan, 1978). A sample of motility-estimated neat semen is exposed to 0°C for about 5 minutes, rapidly warmed to 37°C and then examined for decline in motility and viability. In parts of the world where chilled semen is used for Al, such tests are still in use and help in predicting the survivability of spermatozoa at 4°C during storage. Thomas and Garner (1994) used a combination of fluorescent probes to label fresh and frozen-thawed bull spermatozoa in a flow cytometric evaluation and reported that flow cytometric evaluation of fresh semen may be useful for identifying young sires with relatively poor fertilizing potential. Currently, researchers at the University of Guelph, Ontario, Canada, are working on the possibility of identifying specific enzymes that may be damaged during cryopreservation, and the loss of such enzymes and its effect on fertility. Preliminary reports (Buhr and Zhao, 1995) indicate that Mg2+-ATPase, Na +-K +-ATPase and Ca2+-ATPase are affected during cryopreservation of bull spermatozoa. It has been suggested that such loss or damage of enzymes during cryopreservation may contribute to the reduced fertilizing ability. 29 2.5.4. Acrosome reaction and fertility Saacke and White (1972) found a positive correlation between the percentage of intact acrosomes and non-returns to first insemination, whereas only a weak correlation was obtained when motility estimates were compared with non-returns. Following this report, several workers have examined the relationship between fertility and either acrosomal-integrity or the ability of spermatozoa to undergo acrosome reaction under in vitro conditions. There is strong evidence to show that the ability of spermatozoa to acrosome-react under the influence of heparin or other agents like calcium ionophore A23187 or lysophosphatidylcholine has a definite relationship to fertility (Ax et al., 1985; Parrish et al., 1985; Ax and Lenz, 1986; Graham and Foote 1987a; 1987b; Whitfield and Parkinson, 1992). A relationship between the binding affinity of heparin to spermatozoa and fertility has also been demonstrated (Marks and Ax, 1985; Lalich et al., 1989; Bellin et al., 1993). A test based on calcium ionophore-induced acrosome reaction in human spermatozoa has been found useful in identifying semen samples of subfertile/infertile men, indicating acrosomal dysfunction as a likely cause of fertilization failure. Cummins et al. (1991) have shown this test to have a predictive value for fertility. Results of these studies strongly suggest that the ability of spermatozoa to undergo acrosome reaction in vitro may be useful in predicting the fertility of bulls. 2.5.5. Sperm-zona binding/oocyte penetration assays Sperm-zona binding or oocyte penetration assays (Bosquet et al., 1983; Boatman et al., 1988; Wheeler and Seidel, 1987; Graham and Foote, 1987a; 1987b; Fazeli et al., 1993; Fazeli et al., 1995) have been developed and examined as predictors of fertility. The zona-free hamster egg penetration assay originally described by Yanagimachi (1972) has found some application as a fertility test for use in humans. However, since this assay does not measure the ability of 30 sperm to penetrate the zona, it does not always correlate well with fertilization in vitro (Bosquet et al., 1983; Critser and Noiles, 1993). More recently, Burkman et al., (1988) developed the hemizona binding assay mainly as a diagnostic test for fertility prediction in humans. Since zona pellucida of individual oocytes could vary tremendously in their sperm binding capacity, two matching halves of zona from the same oocyte allows a minimization of such variation. The usefulness of this method for assessing fertility of boar semen has been reported (Fazeli et al., 1995). 2.5.6. Correlation between in vitro and in vivo fertility The birth of the first calf following in vitro fertilization (IVF) was reported by Brackett et al. (1982). Since then, the potential applications of bovine IVF has generated tremendous interest in this technology. As a result, though with limited success, bovine IVF has now become a feasible technology for the production of embryos both for research and commercial applications (Gordon and Lu, 1990; Trounson, 1992). Attempts have been made to correlate the results of bovine IVF with in vivo fertility based on 60-90 d non-return-rates, but conflicting results have been obtained (Oghoda et al., 1988; Hillery et al., 1990; Marquant-Le Guienne et al., 1990). Non-return-rate for a given bull is defined as that percentage of females not returning to estrus within a given period of time (usually 60-90 d) after being bred with semen of that bull. Thus, higher the non-return-rate, better the fertility of the bull in question. In a recent study, Shamsuddin and Larsson (1993) found that 56-60 d non-return-rates were significantly correlated with the first cleavage in vitro, but further embryonic development in vitro was not correlated with non-return-rate. Studies conducted during the course of this thesis project and parallel investigations by others in this laboratory suggest no correlation between IVF and non-return-rates (chapter 4). Further 31 investigations are definitely needed to determine if results of IVF would qualify as a method to predict the in vivo fertility of bulls. From what has been reviewed so far, it becomes apparent that among the various methods available, the most promising approach for predicting fertility at the present time is, perhaps, the assessment of the ability of spermatozoa to undergo acrosome reaction in vitro. Since capacitation is a pre-requisite for acrosome reaction, it will be of interest to assess sperm surface changes associated with capacitation and attempt to correlate such changes with fertility. A better understanding of the processes of bovine sperm capacitation and acrosome reaction in vitro may pave way for developing methods to enhance the success rates in bovine IVF and embryo production systems. 2.6. Techniques for assessing sperm surface changes As discussed earlier, reliable methods for the assessment of sperm surface changes associated with capacitation are unavailable. The acrosome reaction, on the other hand, is relatively simple to identify and several reliable techniques are available. Methods for testing the membrane-integrity of spermatozoa subjected to cryopreservation are currently being developed. 2.6.1. Assessment of sperm capacitation Since no visible morphological changes are known to occur during sperm capacitation, methods for the direct assessment of capacitation are not available. Therefore, assessment of sperm capacitation has been based on indirect measures such as hyperactivated motility (Yanagimachi, 1988; Mcnutt, 1990) and the ability of sperm to penetrate homologous zona pellucida (Wheeler and Seidel, 1987; Boatman et al., 1988). Attempts have been made for 32 developing more objective methods like fluorescent labelling (Byrd, 1981) and radiolabelling (Oliphant and Singhas, 1979) of sperm surface proteins. Many studies have employed lectins for assessing sperm surface modifications associated with capacitation (Koehler and Sato, 1978; Schwarz and Koehler, 1979; Byrd, 1981; Talbot and Chacon, 1981a; Yanagimachi, 1981; Ward and Storey, 1984). Schwarz and Koehler (1979) observed a significant decrease in concanavalin-A and wheat germ agglutinin binding over the equatorial segment and medial regions of the acrosome, after a calcium stimulated acrosome reaction in guinea-pig spermatozoa. The differences in the binding of lectins to capacitated and uncapacitated spermatozoa suggest that the removal of surface coating molecules during capacitation may be responsible for the modifications in lectin binding sites. The use of chlortetracycline (CTC) as a probe to assess sperm capacitation has been reported in several species including the murine (Ward and Storey, 1984; Fraser, 1990; Fraser and Herod, 1990) human (Lee et al., 1987) equine (Varner et al., 1992; Ellington et al., 1993) and the bovine (Byrd, 1981). The CTC is capable of permeating biological membranes to bind with Ca 2 + (Caswell, 1972). The use of CTC as a fluorescent probe to evaluate sperm capacitation seems logical, given its ability to chelate Ca 2 +, and the crucial role this ion plays in these processes. Degelos et al. (1994) used Nanovid microscopy, a technique that allows visualization of large (20-40 nm diameter) particles of colloidal-gold as dynamic markers at the resolution level of the light microscope, to assess sperm membrane changes associated with capacitation and acrosome reaction in bovine spermatozoa. More detailed investigations are needed to establish the practicality of using such techniques. A large number of reports are now available on the potential uses of anti-sperm mAbs to detect sperm surface changes associated with capacitation in vitro. One of the early reports 33 on the use of mAbs to detect sperm capacitation changes was by Menge et al. (1983) who found that their mAbs 1A5 and 22B1 stained the SPM of human spermatozoa incubated in a capacitation medium, whereas they did not stain freshly ejaculated, washed spermatozoa. Myles and Primakoff (1984) found that surface antigens of guinea pig spermatozoa migrate to new locations during capacitation. Okabe et al. (1986) reported that an anti-sperm mAb that they developed was able to identify the capacitation-related disappearance of an antigen from the anterior head region of mouse spermatozoa. The same authors (Okabe et al., 1987) demonstrated the Ca 2 + dependent reactivity of a mAb to capacitated mouse sperm heads. More recently, Fann and Lee (1992), Liu et al. (1992) and Archibong et al. (1995) have found that anti-human sperm mAbs could be useful for detecting capacitation related changes in mouse and human spermatozoa. 2.6.2. Assessment of acrosome reaction Electron microscopy is one of the early methods used for assessing acrosomal status. This method is still considered to be the reference-standard, but is time consuming, expensive and does not allow the differentiation between true acrosome reaction and acrosomal shedding by moribund cells (Critser and Noiles, 1993). Conventional stains such as Giemsa (Saacke, 1970) have worked quite well for assessing acrosome status in bull spermatozoa. Methods have been described to differentiate between live and dead spermatozoa and simultaneously assess the acrosomal status (Aalseth and Saacke, 1986; Didion et al., 1989; de Leeuw et al., 1991; Kovacs and Foote, 1992; Sidhu et al., 1992). A triple stain has been developed for use with human sperm (Talbot and Chacon, 1981b) using a combination of trypan blue, rose bengal and Bismark brown. Though useful, this method is quite time consuming. Very recently, Way et al. (1995) compared four staining procedures, Fast green FCF/eosin-B, eosin-B/aniline blue, trypan 34 blue/Giemsa, and propidium iodide/Pisum sativum agglutinin (PSA), for simultaneous assessment of viability and acrosome integrity of bovine spermatozoa, and reported that Fast green/eosin B, eosin-B/aniline blue and propidium iodide/PSA provided an accurate assessment of both viability and acrosomal integrity of ejaculated bull spermatozoa. In some species, (e.g. guinea-pig and hamster) due to the large size of their acrosomes, phase-contrast or differential interference contrast (DIC) microscopy can be used to easily detect acrosomal status of motile sperm. These methods can also be applied to assess acrosomal status of bull sperm with some difficulty (Saacke and Marshall, 1968; Aalseth and Saacke, 1986), but cannot be used for assessing human sperm acrosome status (Cross and Meizel, 1989). Other methods that have been used for assessing acrosome status include fluoresceinated lectins (Cross et al., 1986; Cross and Watson, 1994), CTC probes (Saling and Storey, 1979; Varner et al., 1987; 1992; Ellington et al., 1993), and mAbs (Wolf et al., 1983; Cross and Meizel, 1989; Fann and Lee, 1992). When conjugated with a fluorescent label, anti-sperm mAbs specific to intra-acrosomal antigens generally show positive acrosomal staining in methanol-fixed acrosome-non-reacted spermatozoa. Upon acrosome reaction, the antibody-binding is lost, as the acrosomal contents would have been shed. There are mAbs such as S-60 and S-74 which are specific to the intra-acrosomal membrane. These mAbs show increased binding to acrosome-reacted sperm, as the IAM will be exposed only after acrosome reaction (Fichorova and Anderson, 1991). 2.6.3. Assessment of cryodamage It has been suggested that reduced sperm survival in frozen-thawed semen is related to membrane destabilization (Foote and Parks, 1993). Sperm membrane integrity and post-thaw motility are considered useful parameters for estimating the cryodamage in spermatozoa. 35 Valcarcel et al. (1994) used the fluorescent probes 6-carboxy-fluorescein diacetate and propidium iodide in fresh and frozen-thawed ram spermatozoa and found the method useful in predicting ram sperm quality and post-thaw survival. The application of an acrosome-specific-mAb in assessing membrane integrity of cryopreserved stallion spermatozoa has been reported (Blach et al., 1988; 1989). For the accurate determinations of membrane integrity in bull spermatozoa, electron microscopy has been used (Krogenoes et al., 1994). Baccetti et al. (1992) used a panel of mAbs to reveal the presence and distribution of the enzymatic and skeletal proteins involved in sperm pathology. They found this approach rewarding in the identification of changes in the antigen localization of bull spermatozoa during epididymal transport, and in the ability to reveal damages caused by anomalies or poor cryopreservation, and suggested that the assembly of a kit of molecular probes to detect bovine sperm integrity would be of advantage. Thus, it is apparent that mAbs are potential candidates for investigating sperm surface changes associated with capacitation, acrosome reaction and cryopreservation in vitro. 2.7. Monoclonal antibodies When a foreign antigen is introduced into the system of a host organism, its immune system is activated, resulting in the production of numerous antibody molecules. Such immunological response to an antigen is heterogeneous in nature. The antibody-secreting cells are referred to as B-lymphocytes; a single activated B-lymphocyte synthesizes antibodies of a particular specificity (Peters, 1992). In the mid-seventies, Kohler and Milstein (1975; 1976) successfully applied somatic cell hybridization and cloning to generate hybrid cell lines producing antibodies with predefined antigenic specificities. They demonstrated that if an antibody-secreting B-lymphocyte is chemically fused to a malignant lymphocyte (myeloma cell), the resultant hybrid cells (hybridomas), will continuously secrete specific mAbs. Myeloma cells 36 are derived from a mutant cell line from a tumour of B-lymphocytes and are virtually immortal. As a result of the mutation, the myeloma cells lose their ability to produce immunoglobulin molecules. Since hybridomas are "clones" of a single hybrid cell, they secrete antibodies of identical nature, acquiring the name "monoclonal antibodies". The unique nature of mAbs is that they are highly antigen-specific. In contrast to polyclonal antibodies, present in whole antisera, mAbs recognize not only a given protein, but one specific site on that protein (Bowen, 1986). The hybridomas are maintained in vitro and will continuously secrete mAbs with a defined specificity. As a result of the B-lymphocytes, hybridized cells convey the specific antibody. In addition, due to the myeloma cells, hybridized cells have the ability to multiply indefinitely in culture. The hybridomas are grown on a selective growth medium known as hypoxanthine-aminopterin-thymidine (HAT). Aminopterin is an inhibitor that blocks the normal biosynthetic pathways by which nucleotides are made. The myeloma cells used for hybridization lack a purine salvage enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT), which results in a defect in the alternate pathway to synthesize nucleic acids (Peters and Gieseler, 1992). The result is that the unfused myeloma cells will die in HAT medium. The unfused spleen cells would die naturally in culture in less than two weeks. Thus, even though spleen cells and myeloma cells would die on their own in HAT medium, the fused cells can survive. The B-lymphocytes provide the enzyme for metabolic bypass, and the myeloma cells are immortal. The hybridomas are cultured as individual clones, each providing a source of mAb. The hybrids are screened for production of specific antibody, and those producing the desired antibodies are chosen for propagation. 37 2.7.1. Applications of monoclonal antibodies Numerous biological applications of mAbs have been reported in the past twenty years. Researchers have been encouraged by the tremendous potential mAbs hold, as tools for specific investigations in several fields including endocrinology, immunogenetics, developmental and cellular biology, reproductive physiology and pathology. The significance of mAbs, and their advantages and disadvantages in comparison to conventional antisera, have been highlighted by Peters and Baron (1992). The applications of mAbs as probes of reproductive mechanisms have been reviewed by Bellve and Moss (1983). 2.7.2. Why monoclonal antibodies were chosen for use in this study Molecular probes specifically meant for assessing bull sperm surface changes are currently not available. Considering the magnitude and importance of the bovine Al industry, and the lack of an understanding of the sperm surface modifications during capacitation, acrosome reaction and cryopreservation, there is a need for the development of suitable biological markers. Since mAbs are highly specific to antigenic determinants, they can be used to investigate changes in proteins of defined specificity with a high degree of precision and repeatability. Numerous studies, in other species, have shown that anti-sperm mAbs are extremely useful to target and study specific sperm antigens. Okabe et al. (1987) using an anti-sperm mAb, demonstrated that capacitation related changes in antigen distribution on mouse sperm heads was directly correlated with fertilization rate in vitro. Fann and Lee (1992) showed that a panel of anti-human and anti-mouse sperm mAbs was useful for the detection of capacitation related changes and acrosome status in mouse spermatozoa cultured in vitro. 38 Since the role of bovine sperm antigens in fertility regulation has not been investigated so far, and due to the non-availability of specific tools to investigate bovine sperm surface changes, it was decided to evaluate anti-sperm mAbs as possible candidates for these purposes. 39 V CHAPTER 3 IDENTIFICATION OF ANTI-HUMAN SPERM MONOCLONAL ANTIBODIES CROSSREACTIVE TO BULL SPERM ANTIGENS 3.1. ABSTRACT In the first experiment, the crossreactivity between bull spermatozoa, and monoclonal antibodies (mAbs) raised against mouse spermatozoa and human spermatozoa, was tested by indirect immunofluorescent assay (IIFA). All the three anti-human sperm mAbs examined (HS-9, HS-11, HS-63) crossreacted with methanol-fixed bull spermatozoa, whereas the anti-mouse sperm mAbs (MS-4 and MS-7) did not. The second experiment (part I) was conducted to determine the binding ability of HS-9, HS-11 and HS-63 with live (unfrozen) bull spermatozoa of 3 bulls incubated (39°C) in a capacitation medium (H-TALP) and to evaluate the three anti-human sperm mAbs as markers for assessing capacitation. The binding of the mAbs to antigens localized in the acrosomal region of live spermatozoa was determined at 0, 2, 4, 6 and 8 h of incubation by IIFA. At the beginning of incubation, binding was minimal (8.9+1.4%, mean ± SE). Maximal binding was observed after incubation for 4 h (51.4±1.6%). In part II, frozen-thawed (bull) and fresh (human) in vitro capacitated spermatozoa were induced to undergo acrosome reaction by treating with lysophosphatidylcholine (bull sperm) or calcium ionophore A23187 (human sperm). The acrosomal integrity of spermatozoa was probed using the mAbs. The mAbs were unable to bind to acrosome-reacted spermatozoa. Similar results were obtained with the three antibodies tested suggesting that these mAbs will be useful markers for the detection of acrosome reaction changes in bull and human spermatozoa. The objective of the third experiment was to purify the cognate bull and human sperm antigen(s) recognized by HS-40 11, to compare their molecular sizes and to examine their immunological relatedness by various immunoassays. By using HS-11 mAb as the affinity ligand, the cognate sperm antigen BSA-11 was purified from bull semen extract by one step immunoaffinity chromatography. Previously purified human sperm antigens HSA-11 and HSA-63 (recognized by the mAbs HS-11 and HS-63) were also used. HSA-11 and HSA-63 consisted of a group of proteins with the same molecular size ranging from 14 to 32 kDa, whereas the bull sperm antigen BSA-11 consisted of only two major proteins of 18 and 20 kDa. The antigens HSA-11, HSA-63 and BSA-11 were immunologically indistinguishable by HS-11 when tested in an enzyme linked immunosorbent assay. In spite of their molecular size heterogeneity, the sperm antigens isolated from the two different mammalian species were commonly recognized by the three independently derived mAbs, HS-9, HS-11 and HS-63. The objective of the fourth experiment was to determine if the mAb HS-11 has any inhibitory effect on bovine sperm-oocyte interaction. A total of 102 oocytes were tested in three replicate trials. No evidence of inhibition of sperm-oocyte interaction was obtained. 3.2. INTRODUCTION The existence of a close antigenic relationship between spermatozoa of different mammalian species was demonstrated in the seventies by several workers (Hansen 1972; Marcus et al., 1973; D'Almeida and Voisin, 1977). These studies, however, were performed using antisera, in which antibodies specific to several antigenic determinants could be present. With the development of the hybridoma technology (Kohler and Milstein, 1975) it is now possible to produce monoclonal antibodies (mAbs) against specific antigens. These antibodies provide highly specific agents that recognize single determinants (Isahakia and Alexander, 1984). The 41 applications of mAbs as molecular probes to understand reproductive mechanisms have been well emphasized by Bellve and Moss (1983). One of the research objectives of our laboratory is to identify or generate anti-sperm mAbs that may prove useful for the in vitro assessment of bull sperm surface changes associated with capacitation, acrosome reaction and cryopreservation. From a panel of anti-human and anti-mouse sperm mAbs previously generated (Lee et al., 1982; 1984b), five mAbs (HS-9, HS-11, HS-63, MS-4 and MS-7) were shown to recognize mouse sperm surface changes associated with capacitation (Fann and Lee, 1992). Since anti-human sperm mAbs have been previously shown to crossreact with sperm antigens of several species including the bovine (Isahakia and Alexander, 1984), it was hypothesized that one or more of the mAbs used in the study by Fann and Lee (1992) may exhibit crossreactivity to bull sperm antigens. This study incorporated four objectives: 1) to determine crossreactivity of the anti-mouse (MS-4, MS-7) and anti-human (HS-9, HS-11, HS-63) sperm mAbs to bull sperm antigens 2) to carry out preliminary evaluation of the crossreacting mAb(s) as markers to assess bull sperm capacitation and acrosome status in vitro 3) to isolate the cognate bull sperm antigen of at least one mAb, and examine its structural and immunological relatedness to its corresponding human sperm antigen and 4) to determine if the selected mAb has any inhibitory effect on bovine sperm-oocyte interaction in an in vitro fertilization (IVF) system. Fulfilment of the second, third and fourth objectives was dependent on obtaining positive results from the first objective. 42 3.3. MATERIALS AND METHODS 3.3.1. Monoclonal antibodies The anti-mouse sperm (MS-4 and MS-7) and anti-human sperm (HS-9, HS-11 and HS-63) monoclonal antibodies previously raised in BALB/c mice were used in this study. The procedures for generation and characterization of monoclonal antibodies against sperm antigens have been reported previously (Lee et al., 1982; 1984a; 1984b). The mAbs HS-11 and HS-63 were selected from more than 100 mAbs generated against human spermatozoa in a single cell fusion experiment, while HS-9 was generated in a separate cell fusion (Menge et al., 1987). All the mAbs including MS-4 and MS-7 (Fann and Lee, 1992) were of the IgGt isotype. 3.3.2. Bull semen Semen samples were collected from Holstein bulls, using artificial vagina, transported to the laboratory at 37-39°C and processed within 1 h. Fresh semen samples pooled from three bulls was used in crossreactivity studies. Single ejaculates of fresh semen from 3 bulls were used in the live sperm-mAb-binding experiment. All semen samples were provided by the British Columbia Artificial Insemination Centre, Milner, British Columbia, Canada. 3.3.3. Determination of the crossreactivity of mAbs to methanol-fixed bull spermatozoa In this experiment, the anti-mouse and anti-human sperm mAbs were screened for their crossreactivity to methanol-fixed bull sperm. Spermatozoa were washed free of seminal plasma in PBS (pH 7.4) and concentration adjusted to 10 x 106 cells/ml. Twenty /xl drops of this sperm-suspension were placed on teflon-coated multi-spot slides (Fisher Scientific Ltd., Ontario, Canada) and allowed to dry on a 37°C slide warmer. Slides were then immersed in 95-100% methanol for 10 min to ensure permeation of the cell membranes and fixation of spermatozoa 43 to the surface of the glass. Each of the five mAbs (initial concentration = 1 mg/ml) was initially diluted 1 in 100 in PBS + 0.5% BSA. Then, two-fold serial dilutions were made, starting from 1 in 1000 to 1 in 128,000. A 10 fil volume of the mAb at each concentration was applied to designated spots on the slides and incubated at 37°C for 30 min in a dark, humid chamber. An unrelated mAb RP 215, previously shown to be specific only to ovarian cancer cells (Lee et al., 1992), was used as the negative control. The slides were then washed and 10 fil of the secondary antibody, Fluorescein isothiocyanate (FITC) labelled goat anti-mouse IgG + IgM (Sigma, St. Louis, MO, USA), at a 1 in 100 dilution was added for an additional 30 min incubation. Slides were then washed three times and observed under an epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY, USA) equipped with excitation and barrier filters ® of 450-490 nm and 520 nm respectively, after applying coverslip on drops of Permaflour (Lipshaw, Pittsburg, PA, USA), an aqeous mountant. Antibody binding was indicated by a bright green fluorescence of the acrosome region of the sperm. Detailed procedures of this indirect immunofluorescent assay (UFA) have been published (Fichorova and Anderson, 1991; Fann and Lee, 1992). 3.3.4. Assessment of the binding of mAbs to live, fresh bull spermatozoa This experiment examined the ability of the mAbs (HS-9, HS-11 and HS-63) to recognize antigens on fresh live spermatozoa in culture and the time course in antibody-binding (as judged by fluorescent staining) to bull spermatozoa. Semen samples of 3 bulls were washed three times in modified Tyrode's medium, (Sp-TALP) pH 7.4 (Parrish et al., 1988; Appendix A), spermatozoa resuspended in Tyrode's medium supplemented with 10 jug/ml heparin sulfate (H-TALP; Appendix A). Spermatozoa (5 x 106/ml) were incubated in 90 ul drops (one drop/mAb and control, corresponding to each incubation period) of H-TALP covered with paraffin oil, in 44 a 24-well culture dish (Nunc, Roskilde, Denmark) and incubated at 39°C in a C0 2 incubator. At 0, 2, 4, 6 and 8 h of incubation, 10 id of each mAb at a 1 in 100 dilution (initial antibody concentration = 1 mg/ml) was added to the corresponding droplet and incubated for 30 min. At the end of each incubation period, the microdrop was aspirated, spermatozoa washed free of unbound mAbs by centrifugation, placed on slides, air-dried, methanol-fixed, incubated with secondary antibody for 30 min, and processed for IIFA. Spermatozoa not incubated with the mAbs (control) were also treated similarly, but incubated with FITC-labelled Pisum sativum agglutinin (PSA: Sigma, St. Louis, MO, USA) at 1 in 100 dilution (instead of FITC-labelled secondary antibody), to serve as an indicator for acrosome status. PSA is a plant lectin known to bind to acrosomal contents (Cross et al., 1986; Mendoza et al., 1992) of mammalian spermatozoa. The acrosomal region of acrosome-intact spermatozoa show a bright fluorescent staining with PSA conjugated to FITC, whereas acrosome-reacted spermatozoa show no staining (Cross and Meizel, 1989). Data from all three bulls were pooled for each mAb to assess the time-dependent changes in antibody binding. 3.3.5. Binding of the mAbs after induced-acrosome reaction Separate experiments were conducted to assess the usefulness of the mAbs to determine acrosome status in the bull and human spermatozoa. 3.3.5.1. Bull spermatozoa: Frozen-thawed semen samples of one bull were used. Spermatozoa were swim-up separated in Sp-TALP medium. After centrifugation, spermatozoa were resuspended in H-TALP, concentration adjusted to 5 x 106 sperm/ml, distributed in six round-bottomed wells (3 control and 3 treatment; 200 iil/well) of a microtitre plate and incubated for 4 h (38.5°C; 5% C0 2 in air). At the end of the 4 h incubation period, spermatozoa in 3 wells 45 were treated with 60 ^ g/ml egg yolk lysophosphatidylcholine (LC; Sigma, St. Louis, MO, USA) and further incubated for 30 min. At the end of this period, spermatozoa were aspirated from both control and treated wells, applied on teflon-coated multi-spot slides (Fisher Scientific Co., Ottawa, ON, Canada) air-dried and fixed in 95-100% methanol for 10 min. Following methanol-fixation, the slides were washed with PBS+0.5% BSA, and blocked with the same solution to minimize non-specific binding. Drops (20 (A) of the mAbs HS-9, HS-11 and HS-63 were applied to designated spots (treated group) and allowed 30 min binding time in a dark humid chamber at room temperature. The unrelated mAb RP 215 served as negative control. The slides were washed, incubated for another 30 min with the FITC-labelled secondary antibody, and washed again before examination. The samples of the control group were incubated with FITC-labelled PSA, washed, mounted and examined. 3.3.5.2. Human spermatozoa: Freshly ejaculated semen from seven healthy donor men were used. A motile population of spermatozoa was obtained by swim-up technique in Ham's F-10 medium (Gibco Laboratories, USA). Samples were distributed in duplicate microwells and incubated at 37°C for 4 h. At the end of incubation, spermatozoa in one well were treated with calcium ionophore A 23187 (10 juM) and incubated for a further period of 90 min. At the end of incubation, spermatozoa from both treated and control wells were aspirated, applied on separate slides and processed as described for bull spermatozoa. In this experiment, however, only the mAb HS-11 was tested. As a positive indicator for acrosome status, FITC-labelled PSA was used in representative wells of each slide. All the slides were examined and results recorded by two independent observers. Mean values of the two observations were taken. Spermatozoa with uniform fluorescence of the acrosome region were considered acrosome-intact, while spermatozoa 46 showing patchy and band-like fluorescence pattern or no fluorescence, were considered acrosome-reacted. 3.3.6. Isolation of sperm antigen by immunoaffinity chromatography Among the three anti-human sperm mAbs tested for crossreactivity with bull spermatozoa, HS-63 exhibited a slightly lower intensity of staining. Other than that, no major differences were seen between the three mAbs. Therefore, it would have been acceptable to use any of the three mAbs for all further evaluations. Since the mAb HS-11 was available in plenty at the time of initiation of this experiment, HS-11 was selected for further studies. Bull sperm antigen reactive to the mAb HS-11 was isolated from bull semen extract by immunoaffinity chromatography. Briefly, a 10 ml pooled sample of bull semen containing 7 x 109 sperm was frozen and thawed several times in PBS. Following centrifugation at 20,000 x g (10 min), the clear supernatant was loaded in a 10 ml immunoaffinity column equilibrated with PBS at pH 7.2. The mAb HS-11 was used as the affinity ligand. After loading, the affinity column was washed extensively with PBS containing 1 M NaCl until the optical absorbency at 280 nm decreased to less than 0.005. The affinity-bound bull sperm antigen was eluted biospecifically with 0.1 M glycine HC1, pH 2.8. The optical absorbency at 280 nm was determined for each 2 ml fraction. The human sperm antigens (HSA-11 and HSA-63) immunopurified using HS-11 or HS-63 as the affinity ligands, were provided for this study by Prof. Gregory Lee, Department of Obstetrics and Gynaecology, University of British Columbia. 47 3.3.7. Molecular weight determination of isolated sperm antigens The eluted fractions of each antigen obtained following affinity chromatography were analyzed for their protein content by their absorbency at 280 nm. Fractions having a high protein concentration were pooled and concentrated by ultracentrifugation, and the molecular size range of the purified antigens analyzed by sodium dodecyl sulphate poly acrylamide gel (10%) electrophoresis (SDS-PAGE; Laemmli, 1970) using a mini-PROTEAN II electrophoresis cell (BIO-RAD Laboratories, Hercules, CA, USA). 3.3.8. Enzyme linked immunosorbent assay (ELISA) To demonstrate the specificity and cross reactivity of the mAb HS-11 to affinity-isolated sperm antigens, the human and bull sperm antigens at 1 ng/ml concentration were coated on microtiter wells for ELISA. HS-11 mAb at dilutions of 1:200 to 1:3200 (original concentration = 1 mg/ml) was used to react with the antigens. Goat anti-mouse IgG+IgM labelled with alkaline phosphatase was used as the secondary antibody. P-nitro phenyl phosphate (2.6 mg/ml in 0.05 M Tris base containing 0.15 M NaCl and 2 mM Mg Cl2) served as the substrate. The colour intensity of the enzymatic reaction was determined at 405 nm using a precision microplate reader (Molecular Devices Corp., Menlo Park, CA, USA). 3.3.9. Influence of HS-11 on bovine in vitro fertilization (IVF) To determine if the mAb HS-11 could impair sperm fertilizing ability, its influence was tested in a bovine IVF system. Swim-up separated bull spermatozoa (pooled sample from 3 bulls) were pre-incubated (30 min) in drops of Fert-TALP medium (Parrish et al., 1988; Appendix A) in the presence of the mAb HS-11 (1 /ng/ml final concentration). Bovine oocytes (n=102) matured in vitro for 26 h were then transferred to the sperm droplets for fertilization 48 (Sivakumaran et al., 1993). The control group spermatozoa were treated with normal mouse IgG (litg/ml). After 12 h, the presumptive zygotes were washed to remove loosely adherent spermatozoa and cultured further. Cleavage and further embryonic development were monitored daily for 3-4 days. 3.3.10. Statistical analysis Differences in binding of mAbs and PSA to bull spermatozoa before and after LC treatment at 4 h, were tested using a t test. Differences in binding of the mAbs between one-another and PSA were tested by ANOVA (SAS, 1986). Relationship between HS-9, HS-11, HS-63 and PSA-binding to bull spermatozoa were examined by regression analysis. The independent observations recorded by each observer were used to calculate the correlation coefficient and the best fit line for the regression was drawn in a scatter plot, using a computer graphics package (Slidewrite Plus version 4.10, Advanced Graphics Software, Sunnyvale, CA, USA). Similarly, the correlation coefficient between percent change in HS-11 and PSA binding to human spermatozoa was calculated and a regression line drawn. 3.4. RESULTS 3.4.1. Crossreactivity of mAbs to methanol-fixed bull spermatozoa Among the five mAbs tested, the anti-mouse sperm antibodies (MS-4, MS-7) did not bind to methanol-fixed bull spermatozoa and were therefore considered non-crossreactive. However, all three anti-human sperm mAbs (HS-9, HS-11 and HS-63) bound to bull spermatozoa with detectable levels of fluorescent intensity up to 1 in 16,000 dilution. 49 3.4.2. Binding of mAbs to live fresh bull spermatozoa When HS-9, HS-11 and HS-63 were individually incubated with fresh spermatozoa in micro-drops, their binding to unfixed live spermatozoa was minimal at time zero (Plate 3.1). Binding trend with the three mAbs was similar. Pooled data from all 3 bulls showed that percent binding (mean + SE) at 0 h was 8.9 + 1.4. No increase in binding was seen at 2 h, but time-dependent changes in the binding of the mAbs were seen thereafter, until 8 h of incubation (Figure 3.1). Maximum binding was observed at 4 h (51.4+1.6%; Plate 3.1) and then gradually declined to 31.7±2.3 percent at 8 h. The FITC-PSA staining remained high (about 80%) between 0 h and 4 h incubation. A marginal decrease in PSA staining was seen beyond 4 h, but even at 8 h incubation the binding was reasonably high (67.0+7.7 %). 3.4.3. Binding of mAbs to sperm before and after induced acrosome reaction Following a 30 min LC treatment, a substantial decrease (P<0.01) in antibody-binding to bull spermatozoa was observed with all the mAbs. However, there were no significant differences (P>0.05) in the change in binding percentage either within the three antibodies or between the mAbs and PSA (Figure 3.2). The percent change in the binding of mAbs and the linear relationship existing between one another is shown in Figure 3.3. In human spermatozoa also, there was a statistically significant (P<0.01; r=0.92) relationship between the percent change in HS-11 binding and PSA binding (Figure 3.4), clearly suggesting that the mAb may be as good a marker as PSA itself for acrosome status assessment. 50 Plate 3.1. Time-dependent changes in the binding of the anti-human sperm mAb HS-11 to the acrosome region of live bull spermatozoa, demonstrated by IIFA. FITC-labelled goat anti-mouse IgG+IgM was used as the secondary antibody. A) Binding at 0 h shown by fluorescence microscopy; B) Same field under normal light; C) Binding after 4 h incubation in H - T A L P medium shown by fluorescence microscopy; D) Same field under normal light. 51 loo r 2 4 6 8 10 Incubation time (h) Figure 3.1. Percent binding (mean+SE) of the mAbs HS-9, HS-11 and HS-63 to fresh live bull spermatozoa, as determined by IIFA, when they were co-incubated for 30-min periods at 0, 2, 4, 6 and 8 h of culture in H-TALP medium. The acrosome status of spermatozoa as determined by FITC-labelled PSA after methanol-fixation is also shown (n=3 bulls; data from all 3 bulls pooled for each mAb). 52 Before L C After L C Figure 3.2. Mean (+ SE) percent binding of the three monoclonal antibodies and that of PSA to bull spermatozoa, before and after LC treatment. There was a significant decrease (P<0.01) in the binding of mAbs to spermatozoa following acrosome reaction consequent upon LC treatment. There were no differences (P>0.05) in binding either within the three mAbs or between the mAbs and PSA. 53 ( c ) 70 r 0 0 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 % change (HS-11) Figure 3.3. The similarity in percent change of the binding of the mAbs HS-9, HS-11 and HS-63 between one another is illustrated. The critical value of correlation coefficient (r) for each of the three combinations was 0.85; P<0.05 (a), 0.90; P<0.05 (b) and 0.92; P<0.01 (c). 54 A B C D Plate 3.2. Results of the SDS-PAGE of purified sperm antigens isolated by immunoaffinity chromatography from human and bull semen extract. From left to right, the lanes represent gels of pre-stained BIO-RAD standard (A), HSA-63 isolated from human sperm (B), HSA-11 isolated from human sperm (C) and BSA-11 from bull sperm (D) respectively. Note that HSA-63 and HSA-11 revealed a group of proteins with similar molecular size (14-32 kDa) whereas BSA-11 comprised of only two major proteins with molecular mass of 18 and 20 kDa. 55 ^ 0 10 20 30 40 50 60 70 80 90 100 % change in binding (PSA) Figure 3.4. The similarity in the relationship between HS-11 and PSA in their ability to detect acrosome changes in human spermatozoa. The critical value of r=0.92 (P<0.01). 56 Figure 3.5. Results of ELISA revealing the binding specificity between the mAb HS-11 and the isolated human sperm antigens HSA-11 and HSA-63 and the bull sperm antigen BSA-11 coated on microwells. Initial antibody concentration used = 1 mg/ml. 57 3.4.4. Molecular size distribution of the isolated antigens The human sperm antigens HSA-63 and HSA-11 when analyzed by SDS-PAGE exhibited a similar group of sperm proteins with a molecular size range of 14 to 32 KDa. The results of this analysis are presented in Plate 3.2 (lane B,C). In contrast, the bull sperm antigen that was purified by HS-11 immunoaffinity column consisted of only two major proteins with molecular mass of about 18 and 20 KDa (Plate 3.2, lane D). 3.4.5. Immunospecificity of the purified antigens The binding specificity between HS-11 and the purified human sperm antigens HSA-11, HSA-63 and the bull sperm antigen BSA-11, was demonstrated by ELISA (Figure 3.5). The immunological relationship between HSA-11, HSA-63 and BSA-11 was also demonstrated by using a sandwich enzyme immunoassay (SEIA) and a competitive enzyme immunoassay (CEIA). Results are not presented in detail. 3.4.6. Influence of HS-11 on fertilization in vitro There was no evidence of inhibition of sperm-oocyte interaction by HS-11 in the IVF system tested. Results are summarized in Table 3.1. Table 3.1. The influence of HS-11 and normal mouse IgG on cleavage of bovine oocytes Trial # mAb HS-11 Normal mouse IgG Oocytes Cleaved Uncleaved Oocytes Cleaved Uncleaved 1 8 3 5 6 1 5 2 21 9 12 21 7 14 3 23 9 14 23 7 16 Total(%) 52(100) 21(40) 31(60) 50(100) 15(30) 35(70) 58 3.5. DISCUSSION The primary purpose of this study was to screen a panel of five mAbs for their crossreactivity with bull sperm antigens as a first step towards identifying potential biological marker(s) to study bull sperm surface changes in vitro. If biological markers are to be used to assess surface changes on unfixed spermatozoa, they must have the ability to react with either sperm surface antigens located on the plasma membrane, or should be able to gain access to internal antigens (located on the outer acrosome membrane, acrosome contents or inner acrosome membrane) of living spermatozoa. Anti-mouse sperm mAbs tested were found non-reactive to bull sperm antigens, whereas, all three anti-human sperm mAbs exhibited strong crossreactivity with methanol-fixed bull spermatozoa, indicating the existence of conserved antigens between the two species. Inter-species crossreactivity between anti-human sperm mAbs and spermatozoa of several mammalian species including the bovine has been previously reported (Isahakia and Alexander, 1984). Having identified three mAbs that recognize antigens in methanol-fixed bull spermatozoa, the next immediate objective was to test if the identified mAbs would bind to live (unfixed) spermatozoa. The cognate antigen(s) of the mAbs HS-9, HS-11 and HS-63 are known to be localized in the acrosomal contents of human and mouse spermatozoa (Anderson et al., 1987; Fann and Lee, 1992). The binding of the mAbs HS-9, HS-11 and HS-63 to unfrozen live bull spermatozoa incubated under capacitation conditions was found to increase in a time-dependent manner, suggesting that the specific antigenic determinant(s) with which the mAbs react become accessible to the mAbs in a time-dependent manner, when spermatozoa were incubated under capacitation conditions. Liu et al. (1992) demonstrated that MSA-63, the cognate mouse sperm antigen of HS-63, becomes exposed during capacitation, and that it is subsequently shed upon acrosome reaction. Fann and Lee (1992) used the mAbs HS-9, HS-11 and HS-63 to monitor 59 their binding to live mouse spermatozoa in different physiological states. Their findings were in perfect agreement with that of Liu et al. (1992) with respect to the time-dependent appearance and disappearance of the antigen. The dynamic nature of sperm surface molecules during the capacitation process is well documented (O'Rand, 1979; Myles and Primakoff, 1984; Okabe et al., 1986; Yanagimachi, 1990). The cognate antigens of the mAbs tested in this study are intra-acrosomal in location. At this time, it is not clear how the intra-acrosomal antigens become accessible to the mAbs when spermatozoa are incubated under conditions favouring capacitation. Either, a pool of antigens should become available on the sperm surface, or, the plasma membrane (SPM) and the outer acrosome membrane (OAM) should become modified/permeable to the passage of mAbs in a time-dependent manner, more so with the progression of capacitation changes. The exact mechanism by which the antigens become accessible needs further investigation. There is little information available on the changes in permeability of the OAM, particularly in association with capacitation. However, it is known that during capacitation, the Ca 2 + ions that penetrate the SPM act, possibly with the participation of calmodulin, on both the SPM and the OAM to facilitate fusion between the two membranes. When the membranes are about to fuse with each other, Ca 2 + enters and H + leaves the acrosome matrix (Yanagimachi, 1990). It would not be illogical, therefore, to presume that as capacitation progresses, the permeability of both SPM and OAM would increase to facilitate passage of ions and other molecules, thus preparing the membranes for the eventual fusion and vesiculation resulting in acrosome reaction. From the results of the induced acrosome reaction experiments carried out, it is quite clear that the conserved acrosomal antigens are indeed lost upon acrosome reaction, in both the species studied. The decrease in mAb-binding to spermatozoa, whether human or bull, was 60 found to be consistent after induced acrosome reaction. This finding is in agreement with the findings of Liu et al., (1992) and Fann and Lee (1992) in mouse spermatozoa, suggesting that the mAbs HS-9, HS-11 and HS-63 may be useful markers for determining acrosome status in the species studied. However, the mAbs may not be able to differentiate between true and false (degenerative) acrosome reactions, unless used in conjunction with a reliable vital staining technique. Sperm-specific antigens which react with monoclonal and polyclonal antibodies have been purified and evaluated previously (Goldberg, 1973; Naz et al., 1984; O'Rand et al., 1984; Primakoff et al., 1988) from several mammalian species. When epitope-specific mAbs were used as probes for the analysis of mammalian gamete antigens, inter-species crossreactivity of some antibodies was documented (Isahakia et al., 1984; Saling et al., 1985; Wilkins et al., 1992). A highly conserved sperm acrosome antigen, MSA-63 which reacts with HS-63 mAb has been purified from mouse testes homogenate and characterized by Liu et al. (1989; 1990; 1992). The biochemical and immunological relationship between the HS-11 cognate mouse sperm antigen MSA-11 and MSA-63, has been reported (Lee et al., 1989). In the present study the conserved acrosome antigen BSA-11, recognized by HS-11 was isolated from bull spermatozoa, compared with the HSA-11 and partially characterized. It was observed from preliminary results that all three mAbs exhibited a similar binding profile to bull sperm in a live (unfixed) sample over a time scale. This observation suggested that the three mAbs may react with the same antigenic determinant, prompting us to test this hypothesis by various immunoassays. The results of such analysis carried out in this study indicate that even though the mAbs used were independently derived, they all recognize the same antigenic determinant, which apparently is highly conserved among different mammalian species. 61 Through ELISA and SEIA it has been shown that the HS-11 cognate human and bull sperm antigens HSA-11 and BSA-11, were recognized not only by the mAb HS-11, but also by HS-63 and HS-9. Since the crossreactivity of these mAbs to mouse sperm has been demonstrated previously, it is possible that the antigenic determinant shared by these mAbs is common to different species. When human sperm antigens HSA-11 and HSA-63 were compared, they were indistinguishable both structurally and immunologically as judged from results of SDS-PAGE and SEIA. Furthermore, any combination of pairing among the three mAbs in SEIA resulted in good quantitative determination of the same antigens in purified form from both human and bull spermatozoa. Finally, the results of a CEIA indicated that all three mAbs compete with one another, apparently for the same binding site on the sperm antigens (HSA-11 or BSA-11) coated on wells. No definite inhibitory influence of HS-11 on IVF could be demonstrated. However, the fertilization rates in all three trials in both treated and control groups were lower than normal, with a large number of oocytes remaining uncleaved. As both treated and control groups had received immunoglobulins either in the form of HS-11 or as normal mouse IgG, it is suspected that these immunoglobulins might have contributed to the lower rates of cleavage. However, since a control group with no additives was not included for comparison, the role of the immunoglobulins must remain a speculation. Recently Archibong et al. (1995) reported a significant inhibitory effect of the anti-human sperm mAb HS-63 on primate (human and monkey) sperm-oocyte interaction. Based on hemi-zona binding assays, they found that HS-63 could inhibit monkey and human sperm binding, up to 99% and 85%, respectively, in a concentration dependent manner. Moreover, the inhibition depended on the state of sperm capacitation, with a maximum inhibition effect seen with capacitated spermatozoa. The inhibitory influence of HS-63 on mouse IVF systems was reported 62 earlier by Liu et al. (1989). In the present study, it has been shown that the mAbs HS-11 and HS-63 are immunologically related and tend to recognize the same antigen, under experimental conditions. If both the mAbs recognize the same antigen, it is intriguing why no inhibitory effect was seen on sperm-zona binding or fertilization in the bovine IVF system. There is reason to believe that species differences may be responsible for such variations in mAb-sperm-oocyte interactions. More detailed investigations are essential to further define this phenomenon. 3.6. CONCLUSION The crossreactivity between three independently derived anti-human sperm mAbs and bull sperm antigens was established in this study. All three mAbs possess the potential for use as markers to determine bull sperm surface changes under capacitation conditions and to assess acrosome status in vitro. Due to the conserved nature of the cognate intra-acrosomal antigen, it is likely that these mAbs will find application in other mammalian species as well. 63 CHAPTER 4 EVALUATION OF THE ANTI-HUMAN SPERM MONOCLONAL ANTIBODY HS-11 AS A MARKER TO ASSESS FERTILITY 4.1. ABSTRACT The present study was designed to determine if there is variation in the binding of the anti-human sperm monoclonal antibody (mAb) HS-11 to spermatozoa of different bulls, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro and in vivo fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5 ml straws) from 5 dairy bulls were used in the first trial. In another independent trial, semen samples of three other bulls were tested. Data from all 8 bulls were considered for analysis. Motile spermatozoa separated by swim-up in Sp-TALP medium were incubated in 90 /tl drops of capacitation medium (H-TALP) at 39°C, 5% C0 2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 (10 ul) was added and the mAb-binding was assessed by indirect immuno-fluorescent assay (UFA). In vitro matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization (IVF). Sperm samples of 2 to 3 bulls were used in each trial, until 4 replicates per bull were attained for IVF (n = 100 oocytes/bull) and UFA experiments. Sperm capacitation status was assessed simultaneously by an egg yolk lysophosphatidylcholine- (LC; Sigma, USA) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was greatest at 4 h incubation in 6 out of the 8 bulls. The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4 h time period. Significant (P < 0.01) differences were observed between bulls in the binding of 64 HS-11 to their spermatozoa (range 22.0+8.3% to 51.8±5.2%) at 4 h. Similarly, variations (P<0.05) in cleavage rate were also seen (range 21.5±9.2% to 58.3±6.5%) between bulls. HS-11 binding and cleavage exhibited positive but weak correlation (r=0.43; n=32; P<0.05) when all 8 bulls were considered. Since one bull showed a totally different trend from others, the data were reanalyzed after excluding that bull. This resulted in a stronger (r=0.56; n=28; P<0.01) correlation. The field fertility (90 d non-return rate) of the eight bulls showed no relationship with HS-11 binding or in vitro cleavage. Results of the LC-induced acrosome reaction at 4 h and the linear relationship between HS-11 binding and cleavage rate observed in the present study together suggest that the binding of the mAb HS-11 to bull spermatozoa on a time-dependent manner indicates capacitation changes. It is concluded that 1) between-bull differences exist in HS-11 binding to spermatozoa and in cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with in vitro fertility but not with in vivo fertility based on first service non-return rate. The anti-human sperm mAb HS-11 may be useful for detecting bull sperm capacitation changes in vitro. 4.2. INTRODUCTION A simple and reliable laboratory test to predict the fertility of breeding bulls has long been required for the bovine artificial insemination (Al) industry. Unfortunately, no such test is presently available. Attempts to develop such a test have met with little success. Even though the basic semen characteristics such as concentration, motility, morphology and freezability are important parameters to be considered, they are of limited value in predicting fertility (Salisbury et al., 1978). More detailed analysis like liverdead ratios and enzyme activity have also been studied (Graham and Pace, 1970; Stewart et al., 1972) but these were also unreliable indices of fertility. Tests based on the ability of bovine spermatozoa to penetrate zona free hamster eggs 65 (Graham and Foote 1987a, 1987b) in vitro, as well as intact homologous oocytes (Marquant-Le Guienne et al., 1990) have been tried and found useful to predict fertility of dairy bulls. However, these tests are time consuming and involve complex procedures. Ax and coworkers (1985) reported that the ability to induce acrosome reaction by chondroitin sulfates in bull spermatozoa in vitro, relates highly to non-return rates of dairy bulls. It has also been suggested that the differences in binding affinity of heparin to bull spermatozoa might be used to predict the fertility of dairy bulls (Marks and Ax, 1985). More recently Whitfield and Parkinson (1992) reported that a significant correlation exists between the fertility of bulls (based on 90 d non-return rate) and the ability of their spermatozoa to undergo acrosome reaction in response to heparin treatment. Even though the process of capacitation is still poorly understood, there is general agreement that only capacitated spermatozoa undergo acrosome reaction (Ohzu and Yanagimachi, 1982; Llanos and Meizel, 1983; Parrish et al., 1988). Since capacitation is a prerequisite for acrosome reaction, there must exist a relationship between capacitation rate and fertility. Unfortunately, there is no direct method available to differentiate between capacitated and non-capacitated spermatozoa in vitro. Presently, assessment of capacitation is based on subjective/indirect methods such as hyperactivated motility, induction of acrosome reaction in response to fusogenic agents (Parrish et al., 1988; Guyader and Chupin, 1991), in vitro egg penetration assays (Wheeler and Seidel, 1987; Boatman et al., 1988), in vitro fertilization assays (Marquant-Le Guienne et al., 1990) and sperm-zona binding assays (Fazeli et al., 1993). Membrane modifications and the migration of sperm surface antigens during capacitation, have been demonstrated using monoclonal antibodies (mAbs) in guinea pig (Myles and Primakoff, 1984) mouse (Okabe et al., 1986), pig (Saxena et al., 1986a) and human (Villarroya and Scholler, 1987) spermatozoa. Time-dependent changes in the reactivity of anti-sperm mAbs to mouse (Okabe et al., 1987; Fann and Lee, 1992) 66 and human (Fann and Lee, 1992) sperm antigens in association with in vitro capacitation and acrosome reaction has been reported. Preliminary studies in this project identified three anti-human sperm mAbs that crossreact with bull sperm antigens (chapter 3). The mAbs reacted with bull spermatozoa on a time-dependent manner when spermatozoa were incubated under capacitation conditions in vitro. These observations suggest that it may be possible to identify capacitation or acrosome reaction changes in bull spermatozoa also, by using these mAbs. If an immunofluorescence based capacitation/acrosome reaction assay could be developed for bull spermatozoa, it would be a significant contribution, not only for possible applications in fertility prediction of bulls, but also to further the understanding of the processes of sperm capacitation and acrosome reaction of bovine spermatozoa. Therefore, the objectives of the present study were: 1) to determine if there is variation in the binding of HS-11 to frozen-thawed spermatozoa of different bulls, and 2) to investigate if there is a correlation between HS-11 binding to spermatozoa and the in vitro and in vivo fertility of the bulls tested. 4.3. MATERIALS AND METHODS 4.3.1. Semen Frozen semen samples from five bulls maintained at the British Columbia Artificial Insemination Centre, Milner, British Columbia were procured. The semen sample of each bull was from one collection (usually two successive ejaculates pooled), split and frozen in 0.5 ml straws. Straws were thawed in a 37°C water bath (40 sec). Spermatozoa were then washed and layered under Sp-TALP medium (Appendix A) for a 60 min swim-up (39°C, 5% C0 2 in humidified air) to obtain an active population of spermatozoa as described by Parrish et al., 67 (1986). About 85% initial motility was observed in the post swim-up samples of all bulls. Semen samples of three additional bulls were subjected to the same procedures in an independent study. 4.3.2. Monoclonal antibody HS-11 The anti-human sperm mAb HS-11 chosen for use in this study was one of the several mAbs previously generated against human sperm in a single cell fusion experiment (Menge et al., 1987). In an earlier investigation the crossreactivity between the anti-human sperm mAbs HS-9, HS-11 and HS-63 and bull spermatozoa was tested and established (chapter 3). The mAb HS-11 was of the IgG, subclass and the initial antibody concentration in the ascites fluid was adjusted to 1 mg/ml. The mAb-containing ascites fluid was stored at -20°C. At the start of each experiment, the mAb was diluted 1:100 in PBS supplemented with 0.5% BSA. 4.3.3. Capacitation procedure Post swim-up sperm concentration was adjusted to 2 x 106/ml and incubated under oil in 90 /il drops of capacitation medium (H-TALP; Appendix A) on 24-well culture dishes (Nunc, Denmark) for 0, 2, 4 and 6 h for capacitation. Conditions for incubation remained the same as for swim-up. 4.3.4. Indirect immunofluorescence assay (IIFA) for mAb-binding Sperm samples of two or three bulls were tested simultaneously. The combination of bulls selected during each trial was deliberately varied each time. At 0, 2, 4 and 6 h of incubation, 10 id of the diluted mAb-containing ascites fluid was added to the appropriate well to attain a final HS-11 concentration of approximately 1 iig/ml. Spermatozoa and HS-11 were co-incubated 30-min for binding. At the end of incubation, the contents of the microdrop were 68 aspirated and placed as 10-15 jul droplets on hydrophobic 8-well slides (Fisher Scientific Ltd., Ontario, Canada), spread uniformly to cover the well, and air-dried. Spermatozoa were then fixed (10 min) in 95-100% methanol. Each well was individually washed 3 times with PBS+0.5% BSA and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG+IgM (Sigma, USA) was added at 1:1000 dilution. Slides were then incubated in a dark humid ® chamber at 37°C for 30 min, washed 3 times as before, cover slip mounted on Permaflour (Lipshaw, Pittsburg, PA, USA) and examined under a fluorescence microscope (x400 to xlOOO) using blue filter combination (excitation filter range: 450-490 nm; barrier filter 520 nm). Sperm counts were made on randomly selected fields until over 100 total spermatozoa were counted from each slide. In each field, the number of total spermatozoa was initially counted under bright field illumination, immediately followed by the counting of FITC-labelled sperm under fluorescent light in dark field to obtain the percent sperm positive for HS-11 binding. The mAb-binding was indicated by a bright green fluorescence of the sperm acrosome region. For assessing acrosomal status of spermatozoa, FITC-labelled pisum sativum agglutinin (PSA; Sigma, St. Louis, MO, USA) was used. Sperm samples were subjected to incubation conditions similar to those used for UFA. Samples were drawn at 0, 2, 4 and 6 h and placed on slides, air-dried and methanol-fixed. A 2 mg/ml solution of fluoresceinated PSA was diluted 1 in 100 and applied to the slides. After incubating for 30 min, excess PSA was removed by washing, coverslip applied on Permafluor and examined under the epifluorescence microscope for fluorescence. 4.3.5. In vitro fertilization (IVF) and assessment of cleavage Procedures for IVF were as reported by Sivakumaran et al. (1993). An illustrative summary of the IVF protocol is presented in Appendix B. Briefly, cumulus-oocyte-complexes 69 aspirated from bovine ovaries collected at slaughter were matured in vitro for 26 h (39°C, 5% C0 2 in air) in the presence of bovine granulosa cells, in Ham's F-10 medium (Gibco, Grand Island, NY, USA) supplemented with 10% estrus cow serum. Good quality mature oocytes were selected and allocated equally to each bull for IVF in Fert-TALP medium (Appendix A). Sperm samples of 2 or 3 bulls were used in each trial on a random basis until each bull had been replicated four times. By the end of the experiment spermatozoa of each bull had been tested against approximately 100 oocytes. The total number of oocytes cleaved by 72 h was recorded separately for each bull. The percent oocytes cleaved at 72 h was expressed as cleavage rate. 4.3.6. Lysophosphatidylcholine- (LC) induced acrosome reaction assay To assess the sperm capacitation status at each incubation period an egg yolk LC (Sigma, St. Louis, MO, USA)-induced acrosome reaction assay was also performed simultaneously. LC was dissolved in ultra-pure water (Milli-Q system) and distributed in 1.5 ml plastic vials (200 /xg per vial), lyophilized and then stored at -80°C until use. Representative samples of spermatozoa cultured for 0, 2, 4 and 6 h in H-TALP were treated with LC (100 /xg/ml) for 15 min. Only capacitated spermatozoa will undergo acrosome reaction in response to LC (Parrish et al., 1988). Treated spermatozoa were then subjected to a dual staining procedure (Trypan blue for viability and Giemsa for acrosome staining). The staining procedure originally described by Sidhu et al. (1992) for detecting true acrosome reaction of the water buffalo spermatozoa was used after minor modifications. Details of the stain composition and modified staining procedure are presented in Appendix C. Four categories of spermatozoa, namely, live acrosome-intact, live acrosome-reacted, dead acrosome-intact and dead acrosome-reacted could be identified without difficulty (Plate 4.1). Live spermatozoa with modified (swollen, discontinuous or denuded) acrosome cap were considered acrosome-reacted. At least 100 live 70 Plate 4.1. Dual stained bull spermatozoa showing the four different states, namely, live acrosome intact (LI), live acrosome reacted (LR), dead acrosome intact (DI) and dead acrosome reacted (DR). 71 spermatozoa were counted from each sample at each of the four time intervals. The percentage increase in acrosome reaction between 0 h and 4 h was chosen for comparisons to avoid the possible confounding effect by spermatozoa undergoing early capacitation and spontaneous acrosome reaction, independent of LC treatment. 4.3.7. Field fertility data The in vivo fertility results of all eight bulls were obtained from the records of the British Columbia Artificial Insemination Centre. Field fertility data for each bull was based on the 90-day non-return rate to first insemination with semen of that particular bull. The number of first services from which non-return rate were collected for each bull varied from 120 up to 3054 over a period of 1 to 3 years. Semen samples used for field inseminations were not from the same ejaculates used for the in vitro studies, even though some inseminations may have been carried out using semen from the same batch. 4.3.8. Statistical analyses Even though two independent studies were taken up, since there was no change in the parameters, data from both the studies were combined and analyzed. Data on percent binding of HS-11 to bull spermatozoa at 4 h, percent increase in LC-induced acrosome reaction between 0 h and 4 h, and the percent cleavage of oocytes assessed at 72 h, were analyzed by analysis of variance (ANOVA) using the General Linear Model procedure of the Statistical Analysis System (SAS, 1986). When the bull effect was significant, mean separation procedure was performed using 't' test. A correlation coefficient between these two parameters was calculated and its significance tested. A linear regression equation was obtained with the data on HS-11 binding and in vitro cleavage rate following IVF. The best-fit line for the regression was drawn in a 72 scatter plot using the Slidewrite Plus (version 4.10) graphics package (Advanced Software Inc., Sunnyvale, CA, USA). The P value of <0.05 was considered significant. 4.4. RESULTS 4.4.1. Binding of the mAb HS-11 The mAb HS-11 showed time-dependent changes in its binding to bull spermatozoa. At the beginning of the incubation, about 20 to 40% binding was seen for sperm samples of all eight bulls, with a gradual increase in the binding percentage as time progressed. The time-dependent changes in HS-11 binding to sperm from 0 h to 6 h was recorded for all the bulls. A maximum binding was observed at 4 h incubation in six out of the eight bulls tested (Figure 4.1). The binding of HS-11 to spermatozoa of two bulls (158 and 230) at 4 h was significantly lower (P<0.01) than that of the six other bulls, with only about 20% sperm showing fluorescent staining at 4 h, compared with 40-50% for the others even though there was no observable difference in motility and viability of spermatozoa between bulls during the various time periods of incubation. On an average, a 30% drop in motility was seen over the 6 h incubation period for all the bulls, but sperm viability remained largely unaffected. 4.4.2. Correlation of HS-11 binding with induced acrosome reaction and in vitro fertility The percentage increase in acrosome reaction of sperm in response to LC treatment after 4 h incubation over values at 0 h for the eight bulls 158, 166, 196, 230, 361, 371, 405 and 438 were 22.5±0.4, 38.5+6.0, 39.0± 18.0, 16.0±2.0, 32.5+3.0, 28.0+8.0, 35.0+3.0 and 26.0±6.0, respectively (Figure 4.2). There were differences (P<0.05) among bulls in HS-11 binding and percentage increase in acrosome reaction after incubation for 4 h. Similarly significant differences (P<0.05) were also observed between bulls in cleavage following IVF 73 (Table 4.1). Binding of HS-11 to bull sperm for the eight bulls and the cleavage rate of oocytes following IVF were significantly correlated (r=0.43; n=32; P < 0.05). It may be observed from Table 4.1 that HS-11 binding at 4 h in seven out of eight bulls was consistently either lower or equal to the cleavage. Spermatozoa of bull 196, however, showed a totally reverse trend with high mAb-binding yet low cleavage. Due to the odd nature of this result, another scatter plot was constructed after excluding the data pertaining to bull 196 (Figure 4.3). This strengthened the correlation to a highly significant level (r=0.56; n=28; P<0.01). Table 4.1. A comparison of between-bull differences in HS-11 binding to spermatozoa at 4 h incubation in capacitation medium, and differences in cleavage rate of in vitro fertilized bovine oocytes. Bull % HS-11 binding % Cleavage #of First service #of it at 4 h (mean±SE) (mean±SE) oocytes non-return (%) Al 158 22.5±4.6b 31.8+4. l"-b 109 76.0 3054 166 43.5±4.5a 43.3+7.8a-b 113 56.7 1032 196 46.3±2.2a 21.5+9.2" 107 77.5 857 230 22.0±8.3b 37.3±7.6"-b 97 70.5 1349 361 51.8±5.2a 58.3+7.4" 101 81.2 120 371 46.0±4.5a 46.3±14.9"-b 108 72.6 200 405 51.0+2.5" 51.8+8.0" 108 59.8 120 438 42.5 + 1.9" 50.5 + 12.0"-b 101 63.4 147 Figures not having a common superscript letter within columns differ significantly (P<0.01 for HS-11 binding; P<0.05 for cleavage). 74 -A— 158 Figure 4.1. The time-dependent changes in mAb HS-11 binding to frozen-thawed sperm of eight different bulls. Mean values of four replicates shown. With the exception of bulls 158 and 230, HS-11 binding to all other bulls was a maximum at 4 h incubation. 75 70 r 6 a o o a 40 o CO O 30 60 50 20 D 10 CO i lLl ih 158 166 196 230 361 371 405 438 B u l l number Figure 4.2. The percent increase in acrosome reaction of sperm from 0 h to 4 h following LC treatment. Note that spermatozoa of bulls 158 and 230 showed the lowest increase in acrosome reaction. 76. fc£ 80 00 70 E-o o 50 40 «4-l o ed 30 2rL 20 10 > - H o U • • • i 10 20 _ i i i _ 30 J i i i i L . 40 50 60 _ i i 70 HS-11 binding to spermatozoa (%) Figure 4.3. The linear relationship between HS-11 binding to spermatozoa and the cleavage rate of bovine oocytes fertilized in vitro after excluding bull 196 (r=0.56; n=28; P<0.01). 77 4.4.3. Correlation with in vivo fertility The field fertility data (based on the 90 d non-return rate to first insemination) of the bulls collected from the records of the Al station are presented in Table 4.1. The data were not correlated either with HS-11 binding or cleavage in vitro, when analyzed statistically. 4.5. DISCUSSION The crossreactivity of the anti-human sperm mAb HS-11 to methanol fixed, as well as fresh live bull spermatozoa was reported in the previous chapter. During initial investigations, it was found that the binding of the mAb HS-11 to spermatozoa varied between bulls. This observation laid the basis for the present study. Earlier studies on the interaction between mAb HS-11 and mouse spermatozoa have shown that HS-11 binds to capacitated, acrosome-intact spermatozoa (Fann and Lee, 1992). Incubation of bull spermatozoa in TALP medium supplemented with heparin at a 10 ttg/ml concentration for a minimum period of 4 h is known to promote capacitation (Parrish et al., 1988). In this study spermatozoa were incubated with 10 /xg/ml heparin for up to 6 h. Significant differences were seen in HS-11 binding to spermatozoa among bulls at 4 h even though sperm motility and viability were not different between bulls. This suggested that capacitation rate of spermatozoa may vary between bulls. Based on HS-11 binding it is apparent that spermatozoa of the bulls 158 and 230 had an inherently low capacitation potential. It was generally observed that bull spermatozoa with low HS-11 binding at 4 h underwent LC-induced acrosome reaction at a lower rate. Their ability to fertilize and initiate cleavage of bovine oocytes was also found to be low. Similarly, spermatozoa with high HS-11 binding showed a relatively higher percentage of acrosome reaction and a proportionately higher ability to initiate cleavage of bovine oocytes in vitro than did spermatozoa with low HS-11 binding. The decline 78 in HS-11 binding by 6 h, suggested that the some of the incubated spermatozoa had started shedding the intra-acrosomal antigen, possibly due to the initiation of spontaneous acrosome reaction. Fann and Lee (1992) reported a similar loss in HS-11 binding after induced acrosome reaction of mouse spermatozoa. All the samples subjected to LC treatment in this study showed a substantial increase in the initiation of acrosome reaction between the 0 h and 4 h time points (Table 4.1). This increase was most likely due to the onset of "true" acrosome reaction. Only dead or moribund sperm could be expected to undergo "false" acrosome reaction. Therefore, LC-induced "false" acrosome reactions would have maximally occurred at 0 h, as little change was evident in the viability of sperm between 0 h and 4 h. This would suggest that the maximum fluorescent staining observed at 4 h may be correlated with the degree of sperm capacitation changes. Reports indicate that there is a direct correlation between fertility rate and the ability of spermatozoa to undergo induced acrosome reaction in vitro (Ax et al., 1985; Whitfield and Parkinson, 1992). Okabe et al. (1987) demonstrated that the reactivity of an anti-sperm mAb with mouse sperm heads on a time-dependent manner and fertilization rate in vitro were directly related. A similar correlation between bovine sperm capacitation (based on HS-11 binding) and in vitro fertility has been established in this study for the first time. Even though a strong relationship existed between HS-11 binding, induced-acrosome reaction and in vitro fertility in 7 of the 8 bulls tested, no such relationship could be found with in vivo (field) fertility. The absence of correlation between in vivo and in vitro fertility of bulls has been found in earlier investigations by Bosquet et al. (1983) and Ohgoda et al. (1988). However, more recently Marquant-Le-Guienne et al. (1990) and Shamsuddin and Larsson (1993) reported that a positive correlation exists between in vitro and in vivo fertility of bulls. As in the present study, Marquant-Le-Guienne et al. (1990) also did not use semen samples of the 79 same ejaculate for in vitro and field trials. As a result, in vivo inseminations were performed from ejaculates which did not necessarily include those that were tested in vitro. Despite this fact, a positive correlation was found in their study. On the contrary, even though Bosquet et al. (1983) and Oghoda et al. (1988) used split samples of the same batch of semen for both in vivo and in vitro inseminations, they could not establish such a relationship. Reports indicate that different lots of semen from the same bull may affect cleavage and embryonic development in vitro (Otoi et al., 1993). To avoid the possible influence of semen-batch differences on the outcome of IVF, split samples from one collection per bull were frozen and used for all IVF experiments in the present study. Since differences in the source and status of eggs can also affect fertilization rates, oocytes from different ovaries were pooled together and selected for maturation and fertilization based on established morphological criteria. Inspite of taking these measures to compensate for the possible variations in sperm and oocytes, no relationship could be found between in vitro and in vivo fertility. There is some evidence to show that male-related embryonic death is not evident at the early cleavage stages, but becomes apparent only after several cleavages (Hillery et al., 1990; Shi et al., 1991). However, this element could not be considered as a source of variation in the present study. Since the actual fertility (pregnancy) rate would be a composite of both fertilization failure and embryonic death, it would perhaps be more appropriate to consider blastocyst formation or hatching of in vitro produced embryos as measures of in vitro fertility. Surprisingly, this study could find no correlation even between induced acrosome reaction and NRR, though others (Ax et al., 1985; Whitfield and Parkinson, 1992) have shown a direct relationship between the two. The absence of a correlation between in vitro cleavage and NRR strongly suggest that variations in the in vitro cleavage of oocytes are independent of NRR following Al when semen samples from the same bull are used. 80 Between-bull differences in the binding of HS-11 to spermatozoa was distinct in the present study. Okabe et al. (1987) also found similar differences from mouse to mouse in the reactivity of their mAb to spermatozoa suggesting that differences in the reactivity of mAbs to spermatozoa, from one animal to another may reflect differences in the ability of spermatozoa to capacitate and fertilize oocytes. FITC-conjugated PSA was used as an indicator for the assessment of acrosomal status. The usefulness of fluoresceinated lectins, including PSA, for assessing acrosome status in bull spermatozoa has been recently described by Cross and Watson (1994). Despite a declining trend, PSA binding remained fairly high even at the 6 h time period in most of the samples tested. Logically, one would expect a proportionate decrease in PSA binding with the progression of acrosome reaction. On acrosomal staining, it was found that even after 6 h incubation under capacitation conditions, followed by exposure to LC, a majority of the live spermatozoa still had their acrosomal cap in place. However, acrosome swelling and discontinuity of membranes were evident in stained samples, suggesting that even though the process of acrosome reaction gets initiated early, shedding of the acrosomal cap is delayed. Since disruption of the external membranes was evident, it is quite likely that portions of the acrosomal contents, including the antigens specific to HS-11, had begun to escape by this time, resulting in the non-availability of the antigens for binding by HS-11 at this time. Probably the remnants of the acrosomal contents still had sufficient glycoproteins to allow PSA binding. This could be the reason for high FITC-PSA staining, yet a low HS-11 binding at 6 h. In summary, time-dependent changes were observed in HS-11 binding to frozen-thawed bull spermatozoa incubated under capacitation conditions, with a maximum binding at 4 h, and a gradual loss of HS-11 binding with the onset of acrosome reaction. Distinct differences existed between bulls in the mAb-binding to their spermatozoa. A close correlation existed 81 between HS-11 binding to spermatozoa at 4 h and cleavage of bovine oocytes (fertilized by spermatozoa of the same ejaculate). The linear relationship between HS-11 binding and in vitro fertility, assessed by the cleavage of bovine oocytes, suggest that the bull sperm antigen recognized by HS-11 may have an important role in the process of fertilization. The present observations suggest that the anti-human sperm mAb HS-11 might be a potential marker for studying bull sperm-surface changes associated with capacitation and acrosome reaction in vitro and for predicting the fertility (in vitro) of bulls, thus allowing the selection of prospective semen donors for bovine IVF programs. Since the binding of HS-11 to fresh (chapter 3) and frozen-thawed spermatozoa (present chapter) was remarkably different, this mAb may also have applications in the assessment of freeze-thaw damage in bull spermatozoa. Further investigations are therefore essential to evaluate the usefulness of this anti-human sperm mAb as a marker for assessing pre-fertilization and post-thaw membrane changes in bull spermatozoa. Since no definite relationship could be established between HS-11 binding and in vivo fertility of the bulls tested, it is doubtful whether this anti-human sperm mAb HS-11 will find application in the prediction of in vivo fertility of breeding bulls. 4.6. CONCLUSION Results of this investigation clearly demonstrate that differences do exist in the binding of the anti-human sperm mAb HS-11 to spermatozoa of different bulls. The observed correlation between induced acrosome reaction, cleavage of bovine oocytes in vitro, and HS-11 binding to spermatozoa incubated under capacitating conditions at 4 h, together strengthen the hypothesis that the maximal binding of HS-11 to bull spermatozoa, is indicative of capacitation changes. Since no correlation could be found between HS-11 binding to spermatozoa and in vivo fertility of the bulls tested, it is concluded that HS-11 may not be a good marker for 82 predicting field fertility of breeding bulls based on laboratory tests. However, HS-11 may be useful for selecting bulls for IVF programs based on the indirect immunofluorescence-based fertility prediction tests. 83 0 CHAPTER 5 PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO BULL SPERMATOZOA 5.1. ABSTRACT The purpose of this study was to raise monoclonal antibodies (mAbs) against bull spermatozoa and to carry out characterization studies on the newly generated mAbs. Six-week-old male BALB/c mice were immunized with PBS-washed whole bull spermatozoa and their spleen cells fused with NS-1 myeloma cells in two separate cell fusion experiments, resulting in the generation of 15 mAbs. The mAbs were specific to antigens of either the posterior tail or the head (intra-acrosomal or plasma membrane) regions of bull spermatozoa. Five major antigenic domains of the bull spermatozoon were identified, corresponding to the five patterns (apical-crescent, equatorial-band, principal-acrosomal, whole-head and posterior-tail) of mAb-binding seen. One mAb specific to the tail region was of IgM class, while the remaining 14 mAbs were of IgG class. They were all sperm-specific with no crossreactivity to bovine oocytes or to any of the twelve somatic tissues tested. However, the mAbs were not specific to bull spermatozoa only, as 11, 10, 2 and 1 of the 15 mAbs crossreacted with sheep, pig, mouse and human spermatozoa respectively. None of the mAbs crossreacted with rooster spermatozoa. The cognate antigens of all the mAbs of the first cell fusion experiment were of testicular origin. However, several of them showed enhanced binding to epididymal spermatozoa. In western blot analysis, 3 of the 13 mAbs tested identified more than one protein band (40 to 200 kDa). Seven others recognized proteins of > 200 kDa, while 3 mAbs recognized none. 84 5.2. INTRODUCTION Monoclonal antibodies (mAbs) have found several applications as probes for understanding reproductive mechanisms (Bellve and Moss, 1983). Anti-sperm mAbs, in particular, are valuable tools for identification, isolation and characterization of surface and sub-surface components of spermatozoa. In recent years, mAbs have been generated against guinea pig (Myles et al., 1981) rabbit (Naz et al., 1983; Lee et al., 1984a) mouse (Bechtol et al., 1979; Feuchter et al., 1981; Schmell et al., 1982), human (Lee et al., 1982; 1984b), pig (Saxena et al., 1986b) and bull (Chakraborty et al., 1985; Bowen, 1986) spermatozoa. Some studies have used mAbs to analyze the developmental expressions of sperm surface antigens during spermatogenesis (Myles and Primakoff, 1983; Lee and Wong, 1986), to demonstrate antigenic regionalization of spermatozoa (Myles et al., 1981; Villarroya and Scholler, 1986) and to study sperm maturation antigens (Eddy et al., 1985). The dynamic nature of sperm antigens and their migration and relocation during capacitation or prior to fertilization have also been demonstrated in laboratory animals (Myles and Primakoff, 1984; Okabe et al., 1986; Fann and Lee, 1992). However, the anti-bull sperm mAbs previously raised have not been tested for their usefulness in understanding sperm surface changes in vitro. Even though bovine sperm physiology has been extensively studied in the past several years, the information available on the distribution of bull sperm antigens and their role in fertilization or on the sperm surface changes associated with capacitation, acrosome reaction and sperm-zona interaction is far from complete. One of the reasons for the dearth of such information may be the non-availability of specific bio-markers. The process of capacitation, first reported over four decades ago by Austin (1951; 1952) and Chang (1951) is still not very well understood. The recent advances in bovine in vitro fertilization (IVF) and sperm-somatic cell co-culture systems have thrown light on some of the mechanisms associated with bull sperm 85 capacitation (Wheeler and Seidel, 1987; Parrish et al., 1988; 1989; 1991; Guyader and Chupin, 1991; Fraser et al., 1995). The availability of specific bio-markers such as anti-bull sperm mAbs may contribute to a more complete understanding of sperm surface changes associated with capacitation, acrosome reaction and sperm-zona interaction. The overall research objective of this series of experiments is to generate immunological markers that may elucidate bull sperm surface changes in vitro, and to correlate such changes with fertility for any possible relationship. Recently we examined the anti-human sperm mAb HS-11 as a potential marker for this purpose (chapter 3 and 4). Even though a correlation was found between HS-11 binding to sperm and fertility of spermatozoa when tested in vitro, no such relationship could be established with in vivo fertility. Since mAbs specifically directed against spermatozoa of homologous species may prove to be more reliable indicators of biological changes, we set out to produce mAbs specific to bull sperm antigens. The characterization of mAbs generated from two independent cell fusion experiments is reported in this chapter. 5.3. MATERIALS AND METHODS 5.3.1. Animals and bull semen Four 6-week-old male BALB/c mice were obtained from the Animal Care Centre, University of British Columbia, Vancouver, and maintained in independent cages according to Canadian Council of Animal Care specifications. Semen was collected by the artificial vagina method from three Holstein bulls (British Columbia Artificial Insemination Centre, Milner, British Columbia) and pooled. The pooled semen was centrifuged once to remove the seminal plasma and washed three times in PBS (pH 7.4) by repeated centrifugation (750 x g for 10 min) and resuspension. The sperm concentration was adjusted to 18 x 106 sperm/ml in PBS and distributed as 400 jul aliquots in microcentrifuge 86 tubes. The sperm samples were either used immediately or stored at -20°C for later injections. Semen samples for characterization studies were used either fresh or frozen-thawed as indicated in the relevant sections of this chapter. 5.3.2. Chemicals and culture media The following items were purchased from GIBCO, Burlington, Ontario, Canada: Ischove's culture medium (IMDM), RPMI 1640 culture medium, fetal bovine serum (FBS), poly-ethylene glycol (PEG 4000). Other components, hypoxanthine-aminopterine-thymidine (HAT; 100 X stock), hypoxanthine-thymidine (HT; 50 X stock), antibiotics (penicillin-streptomycin 100 X) and L-Glutamine (100 X) were obtained from Flow laboratories, Mississauga, Ontario, Canada. Lipopolysaccharide, methylcellulose, dimethylsulfoxide(DMSO), alpha-thioglycerol, Freund's complete and incomplete adjuvants, egg yolk lysophosphatidylcholine (LC), fluorescein isothyocyanate (FITC) conjugated goat anti-mouse IgG+IgA+IgM, horse radish peroxidase (HRP) labelled goat anti-mouse IgG+IgM and poly-L-lysine were purchased from Sigma chemical company, St. Louis, MO, USA. 5.3.3. Immunization and cell fusion Unfrozen sperm suspension emulsified in Freund's complete adjuvant (1:1) was injected subcutaneously (first immunization), 150 /d (1.35 x 106 total sperm) into each mouse in the abdominal region using a 27 G hypodermic needle. The second and third immunizations were done at two-week intervals, using frozen-thawed aliquots of sperm mixed in Freund's incomplete adjuvant at the same concentration as before. Seven days after the third injection, blood samples were drawn from each animal to check for antibody titre in the serum (after ten-fold dilutions from 1 in 10 to 1 in 100,000) by an indirect immunofluorescence assay (IIFA), as described 87 below, using methanol-fixed bull spermatozoa. The antibody titre was determined based on the intensity of fluorescence over the head region of spermatozoa. Based on the antibody titre, one mouse was selected to receive a booster injection of 200 ul sperm in PBS (no adjuvant) into a tail vein using a 30 G hypodermic needle. Three days following the booster injection, the mouse was sacrificed by cervical dislocation and its spleen removed for the first cell fusion experiment. The spleen cells obtained by manual dispersion of the gland were washed and fused with NS-1 myeloma cells at a ratio of about 1:5 in 50 % PEG 4000 and then diluted with serum-free IMDM. The protocol used for cell fusion is presented in Appendix D. Immediately after cell fusion, the mixture of cells was cultured as described by Davis et al. (1982) and Lee et al. (1985a) in a semisolid phase IMDM containing 2% methylcellulose, 25% FBS, 5% HAT stock, lipopolysaccharide (50 Mg/ml), 5 x 106 thymocytes, in 35 mm culture dishes (Appendix D). Seven to 10 days after cell fusion, hybridoma clones that grew as distinct colonies (Plate 5.1) were picked individually under a zoom stereo microscope and cultured in 96-well microtiter plates in IMDM containing 15% fetal bovine serum and HT (no aminopterin). Two days later, culture supernatant was removed from each well and screened for the presence of antibodies that reacted with methanol-fixed bull spermatozoa, by UFA. For the second cell fusion experiment, performed several months later, one of the three remaining mice was sacrificed after a booster injection. The procedures remained essentially the same but for one deviation that live (unfixed) spermatozoa were used for the screening process, mainly to identify mAbs reacting to plasma membrane (surface) antigens. 88 Plate 5.1. Hybridomas in semisolid (methylcellulose) medium 12 days after cell fusion. A) An excellent-growing colony of clones. B) Two smaller colonies in the same culture dish. 89 The positive hybridoma cells from both cell fusions were cultured in RPMI1640 medium containing 10% FBS. On attaining sufficient growth (> 1 x 106 cells/ml) they were frozen in a medium containing 10% DMSO, 40% FBS and 50% IMDM at -80°C overnight and then transferred to liquid nitrogen for storage. Antibody-containing ascites fluid was generated by intraperitoneally injecting pristane primed BALB/c mice with about 5 x 106 hybridoma cells, as described by Lee et al. (1985a). The fluid was drawn periodically by abdominal tap and frozen. Supernatant of the hybridoma culture medium were saved and used in most of the characterization studies. 5.3.4. Indirect immunofluorescence assays (UFA) The UFA procedure was used to screen for the presence of specific antibodies to bull spermatozoa in hybridoma culture supernatant. For screening the hybridoma culture resulting from the first cell fusion experiment, frozen-thawed bull spermatozoa were washed three times in PBS (250 x g, 4 min) and applied to wells of multi-spot hydrophobic glass slides (Fisher scientific, Ontario, Canada) and dried on a warm plate at 37°C. Slides were immersed in 100% methanol for 10 min at room temperature and then blocked (10 min) for non-specific binding using PBS+0.5% BSA. About 20 u\ of the culture supernatant containing mAbs from each hybridoma was placed on fixed spermatozoa and incubated for 30 min in a dark humid chamber at 37°C. The culture supernatant was then removed by aspiration and each spot washed three times with PBS+0.5%BSA, to remove unbound mAbs. Twenty fil of the secondary antibody (FITC-conjugated goat anti-mouse IgG+IgA+IgM at 1:1000 dilution) was placed on each spot and further incubated for 30 min. Following aspiration of the secondary antibody, each spot was washed 3 times, a drop of Permafluor (Lipshaw Immunon, Pittsburg, PA, USA) anti-fade 90 mountant placed before applying the coverslip and examined under an epifluorescence microscope. Excitation and barrier filters with emission wave length of 450-490 nm and 520 nm respectively, were used. For screening, following the second cell fusion experiment, the culture supernatant (75 fil) were distributed in microtitre wells. Live spermatozoa (5 x 106/ml) were added (75 /xl) to each well and incubated for 30 min. The supernatant was discarded after spin-down and the spermatozoa washed 3 times in PBS+0.5%BSA. The final pellet was resuspended, placed on slides pre-coated with poly-L-lysine and incubated with FITC-conjugated secondary antibody, then washed and examined under an epifluorescence microscope. 5.3.5. Tissue and species specificity Frozen tissue sections of bovine lung, liver, heart, brain, pancreas, spleen, kidney, testis, epididymis, ovary, uterus and oviduct were made. The slides were kept at -80°C until use. At the time of use, slides were brought to room temperature, and after blocking for non-specific binding, mAbs in the supernatant were incubated with the tissue sections for 30 min at room temperature. Following three washes with PBS+0.5%BSA, the sections were incubated with FITC-labelled secondary antibody before examination. Bovine oocytes matured in vitro for 24 h were also tested after fixation on poly-L-lysine coated glass slides. To determine if the mAbs were specific to bull spermatozoa only, spermatozoa of human, boar, rooster (ejaculated), sheep and mouse (epididymal) were obtained and tested for crossreactivity by IIFA as described before. Since the anti-human sperm mAb HS-11 was used in previous studies (chapter 3 and 4), HS-11 was also tested for its crossreactivity with other species. RPMI culture medium and non-immunized mouse serum served as negative controls for all IIFAs. 91 5.3.6. Isotyping for class and subclass determination Hybridoma culture medium representing all the mAbs were used in an ELISA system to ® determine the immunoglobulin class and subclass of each mAb. Mouse Typer (BIO-RAD laboratories, Hercules, CA, USA) sub-isotyping kit was used and ELISA was performed according to the manufacturer's instruction manual. At the end of the assay, the microtitre plates were read in a microplate reader (Molecular Devices Corporation, Menlo Park, CA, USA) at 405 nm. The tail-specific mAbs generated in the second cell fusion experiment were excluded from all further characterization studies. 5.3.7. Origin and localization of the cognate antigens To determine whether the sperm antigens recognized by the mAbs are of intratesticular or epididymal origin, bull testicles were collected at slaughter and transported to the laboratory immediately. Epididymal and testicular spermatozoa were obtained and washed in PBS. The sperm concentration was adjusted to about 2 x 106/ml and applied to multi-spot slides, methanol fixed and subjected to UFA with each mAb in duplicate. The mean (percent) binding of mAbs to testicular and epididymal spermatozoa was calculated based on fluorescent stained sperm after counting over 100 spermatozoa in randomly selected fields. Only the mAbs generated from the first cell fusion experiment were tested for the origin of their cognate antigens. To identify the localization sites of the cognate sperm antigens, frozen-thawed live uncapacitated spermatozoa were incubated with each of the mAbs to determine if the antigens were localized on the plasma membrane surface. After incubating sperm in culture supernatant for 30 min, the medium was removed following centrifugation, and the spermatozoa were 92 washed in PBS three times to remove unbound mAbs. Spermatozoa were then coated on slides, methanol fixed and incubated with the fluoresceinated secondary antibody. To determine if the cognate antigens were localized in the acrosome contents, live spermatozoa were washed and air-dried on to multi-spot slides. The slides were then immersed in 100% methanol (10 min) to permeabilize the sperm membranes and expose the acrosomal contents for mAb-binding. UFA was performed as previously described. Acrosome reaction was induced in another sperm population by treating live spermatozoa with LC at 200 ng/ml concentration for at least 8 h to ensure a completely acrosome-reacted population of spermatozoa. After spin-down, the supernatant was discarded, spermatozoa were placed on slides, air-dried and incubated with each mAb of theT BS series (30 min) and then subjected to UFA to determine if any of the mAbs bound to the inner acrosomal membrane (IAM). As it was evident that the mAbs generated in the second cell fusion experiment reacted with surface antigens, live spermatozoa were incubated with either II BS-2 or II BS-11 and colloidal gold-conjugated secondary antibody and for immunoelectron microscopy (IEM) as described in Appendix E. 5.3.8. Western blotting for cognate antigen(s) For identification of the cognate antigen recognized by the mAbs, a 300 ul quantity of pooled bull spermatozoa (1.8 x 109 cells/ml) was sonicated 60 sec on ice with an equal volume of SDS sample buffer. The sonicated sample was then centrifuged (Eppendorf) at 1200 x g for 10 min. The supernatant was removed, j8-mercaptoethanol added (quantity equivalent to 10% of total volume) and boiled for 2 min to achieve reducing conditions. The proteins were then ® separated by SDS-PAGE (7.5 % gel) using a mini PROTEAN-II electrophoresis cell (BIO-RAD 93 Laboratories, Hercules, CA, USA) at 200 V (30-40 min) according to the method of Laemmli (1970). High molecular weight protein standard (BIO-RAD) was used in one lane. The sperm antigens from the gel were then transferred overnight to a polyvinylidine difluoride (PVDF) membrane (BIO-RAD) using a mini PROTEAN-II protein transfer cell, at 30 V. The technique followed was similar to that described for protein transfer to nitrocellulose paper by Towbin et al., (1979). The PVDF membrane was placed in a solution of 5% skim milk in PBS-Tween-20 for 2 h to block non-specific binding sites. Strips (5 mm wide) of PVDF membrane were then cut, labelled and incubated individually with each of the 13 mAbs (undiluted hybridoma culture supernatant) overnight with continuous shaking. At the end of the incubation, the strips were washed 4 times in PBS-Tween-20. HRP-conjugated goat anti-mouse IgG+IgM diluted 1:2000 in PBS+0.5% BSA was used as the secondary antibody for 1-2 h at room temperature and washed thoroughly. In the final step, both the enhanced chemiluminescence (ECL) technique (Amersham UK) and the 3,3'-diaminobenzidine (DAB) methods were used as signal detection systems. 5.4. RESULTS 5.4.1. Hybridoma clones Over 300 hybridoma clones were picked for culture from the first cell fusion experiment. Only those testing positive for antibodies specific to the head region of methanol-fixed sperm were selected for further culture and propagation. At the end of the screening process, 18 hybridoma clones were identified to be secreting mAbs specific to antigen(s) localized in the head region of bull spermatozoa. Eventually, 7 clones failed to continue secretion of mAbs and therefore only 11 could be recovered. The mAbs generated from the first cell fusion were 94 designated as IBS-1,1BS-3, I BS-4,1 BS-5,1BS-6,1BS-8, I BS-9,1BS-10,1BS-11,1BS-12 and I BS-13 (I BS series). Following the second cell fusion experiment, over 800 hybridoma clones were picked for screening against whole live spermatozoa. A total of 24 clones were initially identified secreting mAbs to both surface and internal antigens. Eventually only 4 clones could be salvaged. Two of them continued to secrete mAbs specific to surface antigens localized in the head region, while the other two recognized internal antigens localized in the tail region. The mAbs generated in the second cell fusion were designated as II BS-2, II BS-3, II BS-11 and II BS-19 (II BS series). Cell lines for all the 15 clones were frozen and stored at -135°C. The binding of I BS and II BS series mAbs to bull spermatozoa assessed by UFA indicated five distinct patterns of binding (Plate 5.2). The most frequently encountered pattern was "principal- acrosomal" (9 out of 15). Other patterns included "whole-head" (2 out of 15), "posterior-tail" (2 out of 15), "apical-crescent" and "equatorial-band" (one each). The binding patterns of all 15 mAbs to bull spermatozoa is presented in Plate 5.3 and Table 5.1. Even though the "principal-acrosomal-pattern" was the most common, slight to moderate variations in this pattern could be seen within mAbs (e.g. plate 5.3. D, G and K). Among mAbs of II BS series, II BS-2 showed a diffuse generalized binding over the entire head region (Plate 5.3.L, arrows) of about 50% spermatozoa of the unfixed frozen-thawed semen sample tested. Though II BS-11 also presented a more or less similar pattern, it exhibited a strong affinity to the post-acrosomal region (Plate 5.3.N, arrows). The mAbs II BS-3 and II BS-19 were specific to the posterior tail region, showing no affinity to the mid-piece (Plate 5.3. M and O), and bound to less than 30% of sperm, which was approximately equal to the population of dead spermatozoa, in the semen sample tested. Both the mAbs showed some affinity to the apical head region, but this pattern of binding was not consistently observed. 95 Plate 5.2. Five distinct patterns of mAb binding indicating the five antigenic domains of bull spermatozoa. PA: principal-acrosomal; AC: apical-crescent; WH: whole-head; EB: equatorial-band; PT: posterior-tail. 96 Plate 5.3. Binding patterns of the 15 mAbs to bull spermatozoa as determined by IIFA. I BS series mAbs were incubated with methanol fixed sperm (A-K), while II BS series mAbs were incubated with unfixed live sperm (L-O). Typical acrosomal binding pattern was shown by I BS-1 (A), IBS-5 (D), IBS-6 (E), IBS-8 (F), IBS-9 (G; weak fluorescence), IBS-10 (H), IBS-11 (I), I BS-12 (J) and I BS-13 (K). Crescent shaped binding to anterior acrosomal region (arrows) was shown by I BS-3 (B). The mAb I BS-4 (C) showed strong affinity to the equatorial region (note band-like pattern indicated by arrows). The mAbs II BS-2 (L) and IIBS-11 (N) showed a diffuse but strong binding to the entire head piece; IIBS-11 binding, however, was restricted to the post acrosomal region in some sperm. II BS-3 (M) and II BS-19 (O) recognized antigens located predominantly in the tail region. Only few sperm tails fluorescing since live sperm were used (see materials and methods). Bar = 10 itm. 97 98 5.4.2. Isotype All but one of the 15 mAbs belonged to IgG class. The tail specific mAb II BS-3 was of IgM class. Most mAbs (I BS-4,1 BS-5, I BS-6, I BS-8, I BS-10,1 BS-11,1BS-12,1BS-13 and II BS-19) belonged to the subclass IgGl. II BS-11 belonged to the subclass IgG2a, while I BS-1, I BS-3, I BS-9 and II BS-2 represented IgG2b. 5.4.3. Tissue and species specificity None of the mAbs crossreacted with the twelve different bovine somatic tissues tested. There was no crossreactivity with bovine oocytes either, strongly suggesting that the mAbs are male germ-cell specific. Interspecies crossreactivity was exhibited by most of the mAbs, with sperm antigens of ovine and porcine species being the most commonly recognized. None of the mAbs crossreacted with avian spermatozoa whereas two mAbs recognized murine sperm antigens and one crossreacted with human spermatozoa (Table 5.1). As in the case of II BS-3, HS-11 also crossreacted with spermatozoa of all the species except the avian. The binding patterns of selected mAbs to ovine, porcine and murine spermatozoa are shown in Plate 5.4. In most cases, the cognate sperm antigens of other species recognized by the crossreacting mAbs were localized in the same location as in bull spermatozoa. However, IBS-4 which was specific to the equatorial region in bull spermatozoa, picked-up antigen(s) localized in the equatorial region as well as in the "apical-crescent" in sheep sperm (Plate 5.4.C, arrows). Likewise, HS-11 which consistently showed "principal-acrosomal" binding in all the species, showed a patchy binding to the mid-piece region of pig sperm in addition to the "principal-acrosomal" binding (Plate 5.5). 99 Table 5.1. Interspecies crossreactivity of the anti-bull sperm mAbs mAb # Staining Interspecies crossreactivity to sperm location Bull Sheep Pig Mouse Man Rooster I BS-1 A + + + + + + + + - - -I BS-3 AA + + + + + + - -IBS-4 ER + + + - - - -I BS-5 A + + + + + + + + + - - -I BS-6 A + + + + + + + + + - - -I BS-8 A + + + + + + + + + - - -I BS-9 A + + + - - -I BS-10 A + + + + + - - - -I BS-11 A + + + + + + + + + - - -I BS-12 A + + + + + + + + + - - -I BS-13 A + + - - - - -II BS-2 WH + + + - - - - -II BS-3 T + + + + + + + + + -II BS-11 WH + + - + - - -II BS-19 T + + - - - - -HS-11 A + + + + + + + + + + + + + -Fluorescence intensity: + + + = intense; + 4-= moderate; +=weak; Staining location: A=principal acrosome; AA=anterior acrosome; ER=equatorial region; WH=whole head; T=tail. 5.4.4. Origin and localization of the cognate sperm antigens Results of the IIFAs suggested that the cognate antigen(s) of all mAbs tested (I BS series) were of testicular origin. However, all the mAbs showed enhanced binding to epididymal spermatozoa, with most of the mAbs (I BS-1,1 BS-3, I BS-4,1 BS-8, I BS-9,1 BS-10, I BS-11 and I BS-12) showing a significantly (P>0.05) higher binding (Figure 5.1). The localization of the cognate antigen(s) in spermatozoa was determined by using live uncapacitated, induced acrosome-reacted and methanol-permeated spermatozoa. None of the I BS series mAbs showed significant binding to live membrane-intact spermatozoa. Binding 100 Plate 5.4. Interspecies crossreactivity of selected mAbs as determined by IIFA. Binding patterns of mAbs I-BS-1 (A), I BS-3 (B), I BS-4 (C) and II BS-3 (I) to methanol-fixed sheep sperm. Binding of I BS-6 (D), I BS-3 (E) and II BS-11 (F) to pig sperm, II BS-3 (G) and I BS-3 (H) to mouse sperm. The binding pattern of mAb I BS-3 was similar in all 3 species (B, E, H). The binding of I BS-4 to sheep sperm (C) was slightly different from bull sperm. Apparently the antigens are concentrated in the equatorial and apical regions (arrows). Bar = 10 /xm. 101 Plate 5.5. Patchy binding of mAb HS-11 to mid-piece region of methanol fixed pig spermatozoa, in addition to principal-acrosome staining. HS-11 showed only a principal-acrosome binding in all other species tested. 102 Hi Testicular 1 1 Epididymal 1 3 4 5 6 8 9 10 11 12 13 Monoclonal antibody ( I BS) Figure 5.1. Binding difference of mAbs to spermatozoa of testicular and epididymal origin. Spermatozoa collected from the testicle and epididymis of a bull within 2 h of slaughter were washed in PBS, methanol-fixed and incubated with each of the 11 mAbs of I BS series and processed in duplicate for IIFA. The mAbs I BS-1,1 BS-3,1 BS-4,1BS-9 and IBS-12 showed significantly (*P<0.05) higher binding to epididymal than testicular spermatozoa. 103 occurred to a small population of spermatozoa which was later confirmed to represent dead or damaged acrosome-intact spermatozoa. None of the mAbs bound to induced acrosome-reacted spermatozoa following LC-treatment confirming that the cognate antigens recognized by these mAbs were not localized on the I AM. Since all the mAbs of I BS series showed very high binding to methanol-permeated spermatozoa but not to acrosome-reacted spermatozoa (Figure 5.2), it was concluded that the cognate antigens are all intra-acrosomal. The mAbs of the II BS series that were tested (II BS-2 and II BS-11) readily reacted with a majority of live intact spermatozoa but only showed minimal binding to methanol-fixed spermatozoa (Plate 5.6). The binding of mAb II BS-2 to the sperm surface, confirmed by IEM, is shown in Plate 5.7. Due to the surface-reacting nature of II BS-2 and II BS-11, they were not tested for binding to LC-induced acrosome-reacted spermatozoa. 5.4.5. Molecular size analysis of the cognate antigens Three mAbs of the I BS series identified more than one distinct protein band with molecular size ranges of 40-120 kDa (I BS-3: 6 bands), 70-200 kDa (I BS-10: 5 bands) and 120-200 kDa (I BS-11: 3 bands); Plate 5.8. The mAbs I BS-4 and I BS-5 apparently did not recognize any protein. All the remaining mAbs recognized proteins of > 200 kDa molecular weight. Only two mAbs (II BS-2 and II BS-11) of the II BS series were examined for molecular weight analysis. II BS-2 did not recognize any specific protein while II BS-11 recognized a high molecular weight (— 200 kDa) band. 104 Live L C - A R Fixed Figure 5.2. The general binding trend of the mAbs of I BS-series to live uncapacitated (Live), induced acrosome-reacted (LC-AR) and methanol-fixed (Fixed) spermatozoa. 105 Plate 5.6. Differential binding of the surface reacting mAbs to live and methanol fixed bull sperm as observed by IIFA. The binding of mAb II BS-2 to live (a) and methanol fixed (A) sperm. Similarly, binding of mAb II BS-11 to live (b) and methanol fixed (B) sperm. Bar = 10 itm. 106 1 0 1 1 4 7 5 . 0 kV X i 8 • 0K "l I 6>Vm Plate 5.7. Scanning electron micrograph of a bull sperm showing the localization sites of mAb II BS-2, as detected by colloidal gold (10 nm) labelled goat anti mouse IgG+IgM. Magnification x 18,000. Note tiny white spots (arrows) indicating the sites of antibody localization. 107 it <0 V£) O O O 1-1 oo 10 (N i—i O O w Q o ffl 0) g § 55 | CL , 5 1 •S d 2 H a •> 2 > 3 8 03 -5 B U Ml '3 1/1 (X s • Q w aI ® E I 03 Q S > -9 OH i E <u E o CQ 0) 60 1 m • i — I X O c cL oo OH 0 9 | I t o a o * 8 § § . E &5 « 1 D 52 B § « T 3 en O | M •!?< S Q § 0 0 CL , CO C 3 <yj O 13 i s I I - £ ~ P I J S 3 U H 3 fel I I i I M i * 03 C -3 o oo in PQ V 112 3 108 o r — j E u on OO Ph a) S3 3 X 3 (U B E m Q 0 0 i 03 S "S H 1 x> a 1 to | 8 1 en LH I cx • <L> 0 0 5.5. DISCUSSION In the present study, 15 mAbs against bull spermatozoa were generated and characterized. Anti-bull sperm mAbs have been previously produced (Chakraborty et al., 1985; Bowen, 1986) but no published information is available on their biological applications. Earlier studies in our laboratory using anti-human sperm mAb(s) have demonstrated their potential applications as markers for assessing bull sperm surface changes and suggested a possible relationship between mAb-binding and fertility of bull sperm in vitro (chapter 3 and 4). Since mAbs to homologous spermatozoa may prove to be more specific markers, the purpose of this study was to produce anti-bull sperm mAbs and investigate their usefulness for the aforementioned applications. The activity of mAbs is known to vary dramatically within different assay procedures. For example, Villarroya and Scholler (1986) found that of their 23 anti-human sperm mAbs originally positive in ELISA screening, only 6 bound to spermatozoa when tested by IIFA. Consequently they recommended that if mAbs are raised with the purpose of studying sperm antigen(s) by immunohistological techniques, then an assay such as IIFA should be used for initial screening. Peters and Baron (1992) also emphasized that ELISA should not be used for screening if an immunohistochemical application such as IIFA is proposed. Accordingly, in the present study, IIFA was used for all screening procedures. For screening of mAbs raised in the first cell fusion experiment, we used methanol-fixed spermatozoa. Even though reports indicate minimum changes in antigenicity with methanol- or acetone-fixed spermatozoa (Isahakia and Alexander, 1984; Naz et al., 1984), others have shown that some antigenic determinants may be partly or totally destroyed by standard fixatives (Saxena etal., 1986b; Villarroya and Scholler, 1986). Since we used only methanol-fixed spermatozoa to screen for positive hybridoma clones following the first cell fusion experiment, it is possible that we failed to identify any surface-specific mAbs. The second cell fusion experiment was 109 hence initiated, and live spermatozoa were used to screen for positive hybridomas. This exercise proved rewarding as two surface-specific mAb-secreting hybridomas were identified in the second cell fusion experiment. Of the 15 mAbs raised, in two cell fusion experiments, 11 reacted with intra-acrosomal antigens, two with the plasma membrane of the head and two with antigens localized in the tail region. Isahakia and Alexander (1984) and Villarroya and Scholler (1986) identified 4 and 6 domains of antigen localization, respectively, in human spermatozoa. Five domains have been described for the guinea pig spermatozoa (Primakoff and Myles, 1983) while five classical domains and 16 sub-domains have been described for the domestic pig spermatozoa (Saxena et al., 1986b). Five distinct patterns of antigen localization in the bull spermatozoon have been identified in the present study: a) "apical-crescent-pattern" (crescent-shaped area restricted to the anterior acrosomal region), b) "principal-acrosomal-pattern" (binding to entire acrosome region), c) "whole-head-pattern" (generalized binding to entire head region), d) "equatorial-band-pattern" (restricted collar-type binding to the equatorial region) and e) "posterior-tail-pattern" (binding to the principal piece and end piece of the tail region with no binding to the mid-piece). Based on our observations, it is apparent that, as described for other species, the bovine spermatozoon also has at least five major domains of antigen localization, possibly with several sub-domains. The two distinguishable sub-domains identified during this study were the "post-acrosomal" sub-domain (as seen with the binding of II BS-11 to a sub-population of sperm) and the "incomplete-acrosomal" binding observed with the mAb I BS-9. Even though not all the mAbs were species-specific, they were all sperm-specific with no evidence of crossreactivity with either bovine somatic tissues or oocytes when tested by IIFA. We found that several of our anti-bull sperm mAbs readily recognized sperm antigenic determinants in the domestic livestock species tested (sheep and pig) while the same mAbs 110 showed little or no crossreactivity with human, mouse and rooster sperm antigens. Anti-bull sperm mAbs have previously been shown not to crossreact with human (Chakraborty et al., 1985), mouse (Bowen, 1986) or rabbit (Chakraborty et al., 1985; Bowen, 1986) sperm antigens. Similarly, crossreactivity of anti-bull sperm mAbs with human and mouse sperm antigens was minimal in the present study, with only one and two of the 15 mAbs crossreacting with human and mouse spermatozoa respectively. Inter-species crossreactivity of anti-human sperm mAbs with spermatozoa of different mammalian species has been reported (Isahakia and Alexander, 1984; Shaha et al., 1993). Lee et al. (1984a) demonstrated crossreactivity between anti-rabbit sperm mAbs and human sperm antigens. Isahakia and Alexander (1984) found that only two of the six anti-human sperm mAbs crossreacted with spermatozoa of other mammalian species. Likewise, others (Feuchter et al., 1981; Guant, 1982) have observed crossreactivity of anti-mouse sperm mAbs with sperm of laboratory animals (mouse, rat, rabbit and guinea pig) but not with human spermatozoa. These observations suggest that there may be several sperm antigens commonly shared between closely related species (for instance, ungulates, in the present case), while fewer antigens are shared between distantly related (human, mouse) or unrelated (avian) species. Though mAb-binding was evident to spermatozoa present in cryosections of bovine testis and epididymis, no specific binding of the mAbs was observed to the twelve somatic tissues examined. The binding of mAbs to both testicular and epididymal spermatozoa was further confirmed by UFA using methanol-permeated spermatozoa. Results suggest that the cognate antigens recognized by the mAbs of I BS series are of testicular origin. Interestingly, an enhanced binding (in terms of percent-positive sperm and intensity of fluorescence) of mAbs to epididymal spermatozoa was seen even though the UFA conditions were identical. Moore and Hartman (1984) recorded similar observations in hamster spermatozoa; they found that a mAb 111 (HM 3.1) bound over the head of a higher percentage of cauda-epididymal than testicular spermatozoa and gave a stronger fluorescence on cauda-epididymal than on testicular spermatozoa. Variations in the antigen concentration or localization between testicular and epididymal spermatozoa have also been documented in the rat (Cameo and Blaquier, 1976) and mouse (Guant, 1982). Russell et al. (1984) have shown significant differences in the concentration of major proteins between cauda- and caput-epididymal spermatozoa in the pig, even though the patterns of protein bands appearing in two-dimensional PAGE were remarkably similar. Saxena et al., (1986b) found differences in the pattern of antibody-binding between caput-epididymal, cauda-epididymal and ejaculated pig spermatozoa, when probed with specific mAbs. They observed distinct differences in antigen localization patterns associated with sperm-passage through the epididymis. Changes in the localization patterns, included apparent addition or modification of antigen, loss of antigen and/or reversal of antigen localization from sperm head to tail and vice versa. Drastic changes in the mAb-binding pattern were not observed in the present study, but only an increase in the number of positively stained sperm and intensity of fluorescence were seen between testicular and epididymal spermatozoa. Since protein concentrations between bovine testicular and epididymal spermatozoa were not compared in this study, it is difficult to explain the differences observed. It is likely, though, that an increase in the concentration of intra-acrosomal antigens occurs in the epididymis. Results of our investigations suggested that the mAbs of I BS series do not bind to surface (plasma membrane) antigens of live intact spermatozoa. However, the high positive binding of mAbs to methanol-fixed spermatozoa indicates that the antigens are intra-acrosomally localized and must be exposed (either by methanol-permeation or other means) for the mAbs to access them. No binding was evident after induced acrosome reaction, obviously due to the LC-induced disruption and loss of acrosomal contents. 112 Though both the mAbs of II BS series bound to a majority of live unfixed spermatozoa, the binding was limited to only about 50% (II BS-2) and 30% (II BS-11) of the total spermatozoa in the sample tested. The dead sperm population in the frozen-thawed semen sample used, was approximately 30%. It was tested and found that the mAbs of the II BS series did not bind to dead spermatozoa (killed by allowing to stand at room temperature in TALP medium for over 24 h), possibly due to the loss of surface antigens that may be associated with post-mortem membrane changes. Since the binding of the mAbs was limited only to 30%-50% of an available 70% live sperm population, it is apparent that the mAbs did not bind to all the living spermatozoa. The possibility that the surface antigens were dislodged during the freeze-thaw process, leading to an altered post-thaw antigenicity in 20-40% of spermatozoa cannot be ruled out, as differences in the antigenicity between fresh and frozen-thawed human spermatozoa have been reported by Alexander and Kay (1977), based on antibody-binding studies. On the other hand, it is also possible that spermatozoa present in the semen sample were not homogeneous in their antigenicity. Glassy et al. (1984) identified antigenically distinct subpopulations of human spermatozoa using mAbs. The acrosomal human sperm antigen HS 1A.1 discovered by Villarroya and Scholler (1986) has been shown to be expressed only in a certain percentage of the ejaculated human spermatozoa. It has been suggested that there may exist in the ejaculate various populations of cells at different maturation stages, carrying different antigens on their surface as a result of the continuous modifications of the sperm membrane during spermatogenesis, epididymal maturation or after ejaculation (Bellve and O'Brien, 1983). Another possibility is that some spermatozoa express the relevant determinant at too low a density to be detected by immunocytochemistry (Villarroya and Scholler, 1986). At this stage it is not known if the binding of these mAbs to spermatozoa from different bulls would differ, since semen 113 sample of only one bull was used in the present study. Further studies with semen samples from different bulls may yield some definite answers. The western blot analysis revealed that some mAbs recognized more than one immunoreactive protein band, while three recognized none. It is not unusual for mAbs to detect no band or to recognize more than one during western blot analysis (Primakoff and Myles, 1983; Isahakia and Alexander, 1984; Saxena et al., 1986b; Villarroya and Scholler, 1986). It is possible that the epitopes specific to the mAbs I BS-4, I BS-5 and II BS-2 (that showed no bands) were denatured during solubilization in SDS sample buffer as reported by Thorpe et al. (1984), and failed to restore to the original conformation when transferred to PVDF membrane. Saxena et al. (1986b) suggested that the mAbs detecting multiple bands were probably reactive against determinants of a polymorphic protein species, the components of which might have been disaggregated by SDS during preparation for gel electrophoresis. 5.6. CONCLUSION Fifteen mAbs were generated against bull sperm antigens. All the mAbs were sperm-specific and several of them crossreacted with sperm antigen(s) of other related livestock species. Five distinct antigenic domains of bull spermatozoa have been identified using these mAbs. Further characterization of these newly generated mAbs would be essential to determine their potential biological applications. 114 CHAPTER 6 FUNCTIONAL CHARACTERIZATION AND EVALUATION OF ANTI-BULL SPERM MONOCLONAL ANTIBODIES FOR POTENTIAL BIOLOGICAL APPLICATIONS 6.1. ABSTRACT Four separate experiments were carried out to test the anti-bull sperm mAbs generated in a previous study, for their potential biological applications. Experiment-I was conducted to test their influence on bovine sperm-oocyte interaction in vitro. Eleven mAbs specific to intra-acrosomal antigens and two surface-reacting mAbs were tested in separate sperm-zona pellucida binding assays. A total of 493 oocytes were tested in the presence of the individual mAbs in 3-4 replicate trials. Sperm-zona binding was not affected by 12 of the 13 mAbs tested. However, in the presence of one surface-reacting mAb, the number of spermatozoa bound per zona pellucida significantly increased over untreated control (23.6+5.6 vs 10.0+2.4, mean+SE; P< 0.001). Experiment-II aimed to determine if any of the 10 selected mAbs would detect capacitation related changes of bull spermatozoa in vitro. Frozen-thawed bull spermatozoa were incubated in microdrops of capacitation medium (H-TALP, Appendix A) up to 4.5 h. At 0 h and at 4 h, hybridoma culture supernatant of 10 mAbs were added to individual microdrops (1:1) and incubated with spermatozoa for 30 min. Spermatozoa were then washed to remove unbound mAbs, incubated with FITC labelled secondary antibody and processed for indirect immunofluorescence assay (IIFA). Four of the 8 tested mAbs specific to intra-acrosomal antigens, exhibited a time-dependent increase (P < 0.05) in binding to bull spermatozoa incubated under capacitation conditions. In contrast, the binding of both the mAbs specific to surface antigens significantly decreased (P<0.05) after 4 h incubation in the presence of heparin. The •115 viability of spermatozoa did not change during the 4 h period. In experiment-Ill, the 8 mAbs specific to intra-acrosomal antigens were evaluated as markers to assess bull sperm acrosome status following lysophosphatidylcholine- (LC) induced acrosome reaction. Spermatozoa were incubated in H-TALP, treated with 100itg/ml LC at 0 h and at 4 h, for 15 min, methanol-fixed, incubated with mAbs and the binding of mAbs was assessed by IIFA. FITC-labelled PSA and Giemsa stain were also used as additional methods to verify the acrosome status. A significant decrease (P<0.01) in mAb-binding following induced acrosome reaction was observed with all 8 mAbs which was highly correlated (r>0.85; P<0.01) with PSA and Giemsa staining. The surface-reacting mAbs were not tested in this experiment. In experiment IV, 4 selected mAbs were evaluated as possible indicators of cryodamage in frozen-thawed bull spermatozoa. Fresh and frozen-thawed semen samples of the same ejaculate were incubated with each mAb for 30 min (in duplicate) and processed for IIFA. Ejaculates of five bulls were tested. The binding of mAbs was assessed by two individuals and the means of all four observations were statistically analyzed. No differences were seen between mAbs in their binding to spermatozoa, but between-bull differences were significant. The binding of mAbs to frozen-thawed spermatozoa remained consistently higher, than to fresh spermatozoa. A high correlation (r=0.87; P<0.05) was obtained between mAb-binding and the percentage of dead acrosome-intact spermatozoa. Thus, the hypothesis that the mAbs will bind to live membrane damaged spermatozoa was not supported. Results of these investigations suggest that some of these mAbs are potential bio-markers for bull sperm surface changes associated with capacitation and acrosome reaction in vitro. Apparently one of the surface reacting mAbs promotes sperm-zona binding. 116 6.2. INTRODUCTION Development of laboratory tests to assess semen quality which will accurately predict the performance of bull semen in the field, would provide both livestock breeders and Al organizations with significant economic gains derived from improved breeding efficiency (Saacke, 1970). The importance of laboratory evaluation of bovine semen used for Al has been well emphasized over the years (Salisbury et al., 1978; Saacke, 1982; Hafez, 1987). Despite the availability of standard techniques to assess important semen characteristics such as motility, viability, morphology, acrosomal integrity and enzyme activity, in vitro, no one parameter has been singled out to reliably predict fertility. Even though it is quite unlikely that a simple test or even a series of tests will be all inclusive toward accurate evaluation of semen (Saacke, 1970), it is important to continually look for techniques that may identify new components or characteristics of semen which influence fertility. In their search for more reliable indices of fertility, researchers have recently focused their attention on sperm-oocyte interactions in vitro, as possible predictors of in vivo fertility. Reports are conflicting with some (Marquant-Le Guienne et al., 1990; Fazeli et al., 1993; Shamsuddin and Larsson, 1993) finding a positive relationship between in vitro and in vivo fertility, and others (Bosquet et al., 1983; Oghodaet al., 1988) including our laboratory (chapter 4; Kurtu et al., 1995) finding no such relationship. The existence of a strong linear relationship between the ability of spermatozoa to undergo acrosome reaction in vitro, and non-return rates, has been reported (Ax et al., 1985; Whitfield and Parkinson, 1992). Acrosome reaction is an irreversible exocytotic event, widely occurring in spermatozoa throughout the animal kingdom (Yanagimachi, 1988). The process of capacitation that precedes acrosome reaction, on the other hand, is unique to mammalian 117 spermatozoa and is known to be a reversible phenomenon. Since spermatozoa do not undergo acrosome-reaction unless they have been capacitated, the acrosome-reaction can be used a measure of successful capacitation. However, since unusual incubation conditions or special reagents can forcibly induce acrosome-reaction (Yanagimachi, 1988), the ability of sperm to "acrosome-react" cannot always be accepted as a measure of capacitation or as a fertility indicator. Reliable methods to assess capacitation changes as a fertility indicator may therefore be of significant advantage to the Al industry. The decrease in sperm-viability following cryopreservation causes considerable economic loss to the bovine Al industry. The Al industry is therefore interested in testing cryoprotectants that promise to offer minimal cryodamage to spermatozoa. To effectively carry out such research, simple and efficient techniques are essential for the rapid assessment of cryodamage. The lack of laboratory methodology for detecting sperm cryodamage has been an impediment to sperm cryobiology (Drobnis et al., 1993). Post-thaw motility of spermatozoa has been widely used as measure of membrane integrity by some (Blach et al., 1989; Valcarcel et al., 1994) while others (Krogenoes et al., 1994; Shamsuddin and Rodriguez-Martinez, 1994) have depended on the complex technique of electron microscopy. To enhance our understanding of the sperm membrane modifications during capacitation, acrosome reaction and cryopreservation, and to investigate their importance in fertility regulation, non conventional methods should be examined. In recent years, immunological markers such as anti-sperm monoclonal antibodies have been used to identify and characterize sperm surface changes associated with capacitation in the various mammalian species (Myles and Primakoff, 1984; Okabe et al., 1986; Fann and Lee, 1992; Archibong et al., 1995). In an earlier investigation, the possible relationship between the binding of an anti-human sperm mAb HS-11 to bull spermatozoa incubated under capacitation 118 conditions, and fertility in vitro, was reported (chapter 4). Since a correlation between the mAb-binding and in vivo fertility could not be established, mAbs against bull sperm antigens were generated, hypothesizing that mAbs directed against homologous sperm antigens might be better suited for assessing bull sperm surface changes (chapter 5). The purpose of this study was to perform functional characterization of the newly generated mAbs with particular reference to their involvement in sperm-oocyte interactions and to evaluate them for their potential biological applications. Eleven mAbs (I BS-1,1 BS-3,1BS-4, I BS-5, I BS-6, I BS-8, I BS-9, I BS-10, I BS-11, I BS-12 and I BS-13; I BS series) specific to intra-acrosomal antigens and two mAbs specific to antigens localized in the plasma membrane (II BS-2 and II BS-11; II BS series) were included in this study. Four separate experiments were conducted to a) test the influence of the mAbs on bovine sperm-oocyte interaction in vitro, b) evaluate the mAbs as possible markers for detecting bull sperm surface changes associated with capacitation in vitro, c) examine the possibility of using the mAbs as indicators of acrosome status of bull spermatozoa and d) determine if any on the mAbs would be useful to detect cyrodamage in frozen-thawed bull spermatozoa. 6.3. MATERIALS AND METHODS 6.3.2. Experiment I: Assessment of the influence of mAbs on sperm-oocyte interaction 6.3.2.1. Evaluation of I BS series mAbs To determine if any of the 11 newly generated anti-bull sperm mAbs interfered with the process of sperm-zona binding, homologous sperm-zona binding assays were performed as described by Fazeli et al. (1993) with minor modifications. Bovine oocytes were aspirated from thawed ovaries (frozen within 2 h of slaughter), and used without selection. Methods for recovering oocytes from frozen ovaries for sperm-oocyte interaction studies have been reported 119 (Wheeler and Seidel, 1987; Ellington et al., 1993; Miller and Ax, 1995). Spermatozoa (lOxlO6 cells/ml in H-TALP) and hybridoma culture supernatant (or medium only, as control) were mixed 1:1. The heparin concentration in the medium was adjusted in such a manner to avoid the dilution effect, ensuring a final concentration of 10 tig/ml heparin. Spermatozoa and mAbs were pre-incubated for 30 min at 38.5°C (100% humidity) in individual microtitre wells for the antigen-antibody binding to occur. At the end of this period, oocytes were randomly picked and added to each well at the rate of 5-10 oocytes per well depending on the total number available, and incubated for 4 h. At the end of 4 h, the oocyte-sperm complexes were aspirated in a 100 itl Eppendorf pipette tip and rinsed 10 times (instead of 5, in the original procedure) to remove loosely attached sperm. With 5 rinses, the number of sperm bound to the zona remained high, posing difficulties in counting; therefore 5 additional rinses were done. The washed oocytes were fixed in 2.5% glutaraldehyde (10 min), stained with Hoechst 33342 nuclear stain (1 mg/ml in PBS; Sigma, St. Louis, MO, USA) for 10 min, transferred to glass slides, slightly compressed under a coverslip (corners supported on wax), the edges sealed with nail polish and examined under epifluorescence microscope using Nikon UV-2A filter combination (excitation filter=330-380 nm; barrier filter=420 nm). Sperm bound to the zona pellucida could be easily counted as they were brightly fluorescent (Plate 6.1). In all, 179 oocytes were tested in three separate sperm-zona binding assays to attain a total of 15-20 oocytes for each mAb. 6.3.2.2. Evaluation of II BS series mAbs Two surface-reacting mAbs of II BS series (II BS-2 and II BS-11) were tested for their influence on sperm zona binding. Procedures of the assay remained the same as for I BS series. However, in this case, two controls were used. The positive control was immune mouse serum, collected (and stored until use at -20°C) from the mouse used for raising the antibodies. The 120 negative control was normal (non-immune) mouse serum, both diluted to contain approximately the same protein concentration. About 75 oocytes were tested for each mAb in four replicates (n=314 oocytes). 6.3.3. Experiment II: Evaluation of mAbs as markers for capacitation related surface changes All the eleven mAbs of I BS series, and two mAbs (II BS-2 and II BS-11) of the II BS series were evaluated to determine if any time-dependent changes occurred in their binding to spermatozoa. The mAbs I BS-3, I BS-4 and I BS-9 were eventually excluded from further evaluations due to weak fluorescent signals. Time-dependent changes in mAb-binding were assessed at 0.5 h and 4.5 h. Frozen-thawed spermatozoa (pooled from three bulls) were washed 3 times in Sp-TALP medium and split into two parts. One part was resuspended in the same medium (Sp-TALP, no heparin) and the other part suspended in capacitation medium (H-TALP). The sperm concentration was adjusted to 5 x 106/ml and a 75 id aliquot was distributed to labelled wells of two microtitre plates (earmarked for 0.5 h and 4.5 h). An equal volume of the corresponding hybridoma culture supernatant was then added to each well of the 0.5 h plate. The control well received 75 ill of the culture medium containing no mAbs. The microplate was incubated (38.5°C; 5% C0 2 in air; 100% humidity) for 30 min. At the end of the incubation, spermatozoa were washed three times by centrifugation at 300 x g for 3 min each time, taking care to remove the supernatant after every spin before adding fresh washing medium. After the final rinse, spermatozoa were processed for IIFA. Briefly, spermatozoa were coated on multi-spot slides (Fisher Scientific Ltd., Ontario), methanol-fixed for 10 min and incubated with FITC-labelled goat anti-mouse IgG+IgM+IgA (1:1000 dilution, Sigma, USA) for 30 min at 37°C, 121 Plate 6.1. Sperm-zona binding assay. Note Hoechst 33345 stained spermatozoa bound to the zona pellucida of bovine oocytes (x 400). 122 washed and examined under epifluorescence microscope. The 4.5 h samples were also processed in a similar fashion for IIFA after 30 min incubation with the mAbs added at 4 h. The experiment was repeated four times. At least 200 spermatozoa were counted each time and the percent binding recorded. Spermatozoa with complete, uniform fluorescence over the acrosome cap region were counted as positive, while sperm heads showing a band-type fluorescence on the equatorial region, those showing irregular or patchy fluorescence, and those with no fluorescence were counted as negative. 6.3.4. Experiment HI: Evaluation of mAbs as markers to assess acrosome status The binding of mAbs to bull spermatozoa under conditions favourable for capacitation, and following induced acrosome reaction was determined by IIFA in this experiment. Simultaneously, spermatozoa were also subjected to a dual staining (trypan blue and Giemsa combination; see Appendix C) technique suitable for light microscopy and to a fluorescence microscopy technique using FITC-labelled pisum sativum agglutinin (PSA) to verify the acrosome status (Cross et al., 1986; Mendoza et al., 1992; Cross and Watson, 1994). Stock solutions of lysophosphatidylcholine (LC; 5 mg/ml in methanol) and heparin (1 mg/ml in Sp-TALP medium) were prepared and kept frozen at -80°C. Before starting the experiment, the methanol was evaporated under a stream of nitrogen gas and the LC reconstituted in 2 ml Sp-TALP medium. Frozen semen straws from three different bulls were thawed, the semen pooled together and washed twice in Sp-TALP medium by centrifugation for 3 min at 300 x g, and concentration adjusted to 5 x 106/ml. Heparin was added to the washed sperm suspension in quantity sufficient to attain a concentration of 10 itg/ml. The sperm sample was distributed as 300 til microdrops to 8 wells of a 24-well tissue culture plate labelled as follows: 0.5 h control (for IIFA), 0.5 h LC (for IIFA), 0.5 h control (for dual staining), 0.5 h LC (for dual staining), 123 4.5 h control (for UFA), 4.5 h LC (for UFA), 4.5 h control (for dual staining) and 4.5 h LC (for dual staining). The drops were overlaid with 1 ml of mineral oil. Immediately after distribution, LC was added to the two 0.5 h LC drops to attain the final cone, of 100 ng/ml. All the drops were incubated at 38.5°C, 5% C0 2, 100% humidity. After 15 min, 250 ul of the contents of the four 0.5 h drops were aspirated, mixed with an equal volume of Sp-TALP medium in microfuge tubes and washed by centrifugation to minimize the effect of the left over LC, on sperm. Control tubes were also treated similarly for the sake of uniformity. Aliquots (10 (A) of sperm suspension (0.5 h control and 0.5 h LC) were then quickly distributed to the appropriate spots of labelled multi-spot slides and air dried. The sperm preparations were then fixed in 100% methanol, and processed for UFA after incubating with each of the 8 mAbs of I BS series (undiluted hybridoma culture supernatant or 1:1000 dilution of ascites fluid). After 4 h incubation, LC was added to the two 4.5 h LC drops and continued incubation for 15 min and processed as before. Two spots of a slide (at 0.5 h and at 4.5 h) were saved for FITC-labelled PSA. At the time of adding the secondary antibody, FITC-labelled PSA was applied to the spots allocated for that purpose. The experiment was repeated three times. Spermatozoa showing a complete bright fluorescence of the acrosome region were counted as acrosome-intact. Patchy, partial or irregular fluorescence was indicative of acrosome reacting sperm, while a band-like fluorescence pattern or absence of fluorescence indicated acrosome-reacted spermatozoa (Cross et al., 1986). For convenience of scoring, both acrosome reacting and acrosome-reacted categories were counted together as acrosome-reacted. The difference in the percentage of acrosome-reacted spermatozoa between control and treated groups at any given time reflected the actual percentage of spermatozoa that underwent acrosome reaction due to LC treatment. Even though it is difficult to provide conclusive evidence that this method would help identify sperm that underwent true acrosome reaction, since only the 124 difference between control and LC-treated groups was considered, there is reason to believe that this would have given a reasonable indication of true acrosome reaction. Samples for dual staining were processed in the following manner. After mixing the aspirate (250 id) from the respective drops with equal volume of TALP, the mixture was centrifuged, 450 /xl of the supernatant was removed, leaving the sperm in about 50 til at the bottom. To this, 50 /xl of 0.2% trypan blue solution (prepared in TALP free of BSA and heparin) was added, mixed and set aside for 3 minutes at 38°C. At the end of 3 min, smears were made on clean glass slides and allowed to dry at 45° angle for 15 min before staining with a Giemsa-methanol-glycerol mixture (Appendix C). 6.3.5. Experiment TV: Evaluation of mAbs to assess cryodamage in frozen-thawed sperm The purpose of this experiment was to evaluate the mAbs as potential indicators of cryodamage in frozen-thawed bull spermatozoa. Based on a preliminary trial, four I BS series mAbs were selected for further evaluations. Since it has been suggested that sperm plasma-acrosome membrane integrity is affected during freezing and thawing (Check and Check, 1991; Parks and Graham, 1992; Valcarcel et al., 1994), we hypothesized that our mAbs may readily gain access to their cognate intra-acrosomal antigens in spermatozoa with compromised membrane integrity. After incubation with the primary antibody (mAb), if the FITC-labelled secondary antibody is added, the membrane damaged sperm should fluoresce green when viewed by epifluorescence microscopy. Ejaculates of five different bulls were used in this study. Fresh and frozen semen samples of the same ejaculate from each of the five bulls were tested in duplicate for mAb-binding. Briefly, washed spermatozoa (5 x 106/ml) were incubated in duplicate in PBS + 0.5% BSA with the four mAbs in independent microwells of a 96-well microtiter plate for 30 min at 38.5°C (5% C0 2 in air), centrifuged and washed three times to 125 remove unbound mAbs, fixed on slides and processed for UFA as described before. Fresh semen samples were processed within 3 h of collection, while frozen samples were thawed 1-2 weeks after the freeze date and processed immediately for incubation with mAbs and UFA. Spermatozoa were scored for mAb-binding by two individuals, each counting approximately 300 sperm cells in randomly selected fields. The viability and acrosomal status of spermatozoa of both fresh and frozen-thawed samples were assessed using the dual staining technique of Sidhu et al., 1992 as detailed in Appendix C. The nuclear stains bis-benzimide (Hoechst 33342 and Hoechst 33258; Sigma) were used to test the membrane-integrity of frozen-thawed spermatozoa of two bulls. Sperm samples were incubated for 10 min with the stain (either 0.1 /xg/ml Hoechst 33342 or 0.5 /xg/ml Hoechst 33258) and quickly examined as a wet smear under coverslip in the fluorescence microscope using Nikon UV-2A filter combination. Unstained or partially stained spermatozoa were considered membrane intact. 6.3.6. Statistical analyses Data from the time-dependent binding, induced acrosome reaction, cryodamage and sperm-zona binding assays were tested by ANOVA using the General Linear Models (GLM) procedure of Statistical Analysis Systems Inc. (SAS, 1986). When the model was found significant, differences between means were tested by using either Duncan's Multiple Range Test (time-dependent binding data) or Probability of Difference (sperm-zona binding and cryodamage data) as outlined in the GLM procedures of SAS user's guide. 126 6.4. RESULTS 6.4.1. Influence of mAbs on sperm-oocyte interaction Results of the sperm-zona binding assays provided no evidence of inhibition of the sperm-oocyte interaction by any of the mAbs of I BS series. When data from all 11 mAbs were pooled, an average binding of 14.4+2.0 spermatozoa per ovum (n = 179; mean 16.3 oocytes per mAb) was observed, which was not different (P>0.05) from untreated control (14.9+2.1 spermatozoa per ovum; n = 17). Similarly, the mAb II BS-11 also did not inhibit sperm-zona binding. However, II BS-2 apparently had a positive influence on sperm-zona interaction, in that, sperm incubated with this mAb showed a significantly higher (P< 0.001) binding to zona much greater than with II BS-11, positive and negative controls (Table 6.1; Plate 6.2). Results of the sperm-zona binding (mean+SE sperm/zona) in the presence of the mAb II BS-2, all other mAbs combined, normal mouse serum and immune mouse serum are presented in Figure 6.1. Table 6.1. The influence of mAbs II BS-2 and II BS-11 on sperm-zona interaction mAb # Oocytes used (n) Number of sperm bound per zona (mean±SEM) II BS-2 79 "23.6+5.6 II BS-11 75 "11.9+3.4 IMS 75 c2.9±1.0 NMS 68 "10.0+2.4 IMS = Immune mouse serum (positive control; maximum inhibition expected) NMS= Normal mouse serum (negative control; no inhibition expected) "•"•"Figures with different superscripts differ significantly (P< 0.001) 6.4.2. Assessment of capacitation related changes Among the eight mAbs of I BS series evaluated, four (I BS-1, I BS-5,1 BS-12 and IBS-13) showed increased binding to spermatozoa at 4.5 h when compared with their binding at 0.5 h. Even though a 15-20% drop in sperm motility was observed between 0.5 h and 4.5 h, the 127 viability of sperm did not change during this period. In all four cases, heparin had no influence on mAb-binding. Since heparin effect was not seen both at 0.5 h and at 4.5 h, the data from control and heparin treated groups of the respective time periods were pooled and statistical differences between the pooled 0.5 h and 4.5 h data tested. The differences were significant (P<0.05; Figure 6.2.a). There was no statistical difference (P>0.05) in the binding of the remaining 4 mAbs (I BS-6,1 BS-8,1 BS-10 and I BS-11) between 0.5 h and 4.5 h (Figure 6.2.b) even though the culture and incubation conditions remained identical. The binding percentage (at 0.5 h) of the two mAbs of II BS series, was higher than that of the I BS series mAbs (range: 28.8% to 49.3% vs 2.4% to 26.4% respectively). As observed with the I BS series mAbs, heparin did not significantly influence the binding of II BS-2 or II BS-11 at 0.5 h, though a tendency for lower binding to heparin treated sperm was evident with both the mAb(s) at this period. However, at 4.5 h, the binding of both the mAbs was significantly lower (P<0.05; Figure 6.3) to heparin treated sperm as against the control. 6.4.3. Assessment of acrosome status The binding of all the eight mAbs evaluated, was found to be highly correlated (R>0.85; P<0.01) to PSA staining, when compared individually. No significant differences were seen among binding of mAb(s), PSA and dual staining (Giemsa) techniques when used as methods for identifying acrosome-intact spermatozoa. A large population of acrosome-intact spermatozoa (>70%) was identified alike by the mAbs, PSA and Giemsa stain at 0.5 h. Even though LC treatment did induce acrosome reaction at this period in a small population of spermatozoa, a large percentage of spermatozoa underwent acrosome reaction in response to LC only at 4.5 h (Figure 6.4; Plate 6.3). A high incidence of spontaneous acrosome reaction was observed 128 Figure 6.1. Comparison of mean binding of spermatozoa to oocytes (number bound per zona) after incubation with II BS-2, all other mAbs (mean), normal mouse serum (negative control) and immune mouse serum (positive control). Bars bearing different letters are significantly different (P<0.001). 130 (a) (b) 40 r WD .2 20 1 5 12 13 Monoclonal antibody (I BS) 0.5 h 4.5 h Monoclonal antibody (I BS) Figure 6.2. Time-dependent changes in the binding of 8 mAbs (I BS series) to bull spermatozoa, tested live, under capacitation conditions. Frozen-thawed spermatozoa were washed and incubated either immediately with mAbs for 30 min (0.5 h) or after 4 h incubation in capacitation medium (4.5 h). The experiment was repeated 4 times, (a) The mAbs I BS-1, I BS-5, I BS-12 and I BS-13 showed an increase in binding to sperm incubated for a longer period (P<0.05); (b) The mAbs I BS-6, I BS-8, I BS-10 and I BS-11 showed time-dependent increase in binding. 131 Figure 6.3. Binding of the surface reacting mAbs to live bull spermatozoa incubated under capacitation conditions. At 4.5 h, the binding of II BS-2 and II BS-11 were significantly lower to the heparin treated samples in comparison with heparin-free samples. 132 90 r 0.5hC 0.5hT 4.5h C 4.5h T Time & treatment Figure 6.4. Acrosome status of control and LC treated bull spermatozoa at 0.5 h and at 4.5 h determined by mAbs, FITC-labelled PSA and Giemsa stain. 133 134 between 0.5 h and 4.5 h, among spermatozoa of the control group. This effect, most likely, was due to the influence of heparin present in the culture medium. 6.4.4. Assessment of cryodamage The binding of mAbs to frozen-thawed spermatozoa was always higher than fresh spermatozoa (P < 0.01). This was true for all the five bulls. The mean percent binding of mAbs to spermatozoa varied considerably between bulls (Table 6.2; Figure 6.5). However, when data pertaining to each mAb were pooled from all 5 bulls, closely similar values were obtained for each mAb. The mean (+SE) pooled percent binding of the mAbs I BS-1, I BS-5, I BS-8 and I BS-10 to fresh and frozen-thawed sperm were 15.0±1.3 vs25.8±1.6, 16.2+1.2 vs 24.2+1.3, 14.9+1.6 vs 23.1 ±1.4 and 13.9+1.0 vs 20.1 + 1.4 respectively. Viability and acrosome status estimates obtained by dual staining indicated a relationship between mAb-binding (to both fresh and frozen-thawed sperm) and dead acrosome-non-reacted spermatozoa. On statistical analysis, a close correlation (r=0.86; P<0.01) between the two was established (Figure 6.6). The bis-benzimide stain Hoechst 33342 was found not suitable for assessing membrane integrity of bull spermatozoa, since even at very low concentrations (0.1 /xg/ml), the dye was able to stain close to 100% of the spermatozoa (Plate 6.4). Similarly, Hoechst 33258 also showed high staining, when used at litg/ml, the concentration previously suggested for use on human sperm (Cross et al., 1986). However, by gradually reducing the concentration, 0.5 itg/ml was determined as ideal, since distinctly different populations of sperm could be identified (Plate 6.5). When frozen-thawed sperm of two bulls were stained with Hoechst 33258, in duplicate, an average of 26% and 36% sperm took-up the stain suggesting membrane damage. The corresponding values for mAb-binding to the sperm of these two bulls were 17% and 28%, roughly 10% less binding than to nuclear stain. 135 PQ ^ 1-1 c Id O O w CO gp| PQ .S c c o M| PQ .S FH " O T - H * W PQ .5 >-H c c N s PL, on PH c N o no PH c N s PH PH c N a PH tU T H PH m i n 0 0 0 0 o +1 CN +1 T - H r-" +1 T - H .7+4. CN +1 CN T - H +1 T - H r - H CN c o CN T - H CN © CN © CN C O q v O O O q q 8±0.: c o +1 O N O +1 CN CN +1 CN +1 c o * - H +1 O N n o C O o d c o 0 0 o o N O I > N O 'l+L T - H +1 CN +1 T - H +1, ,0±6. +1 T - H m t - H NO CN ON CN © CN ^ -CN CO CN o o CN N O c o O O N O T - H +1 +1 i n T - H +1 c o CN +1 r -CN +1 T - H +1 ON c o i n CN NO T - H 0 0 O i > CO c o +1 O O +1 CN N O +1 N O CO +1 CN N O +1 q T - H +1 CN p - ' T - H o " CN i n CN O CN CN CN . c o N O m m CN ,2±3. i n +1 wo o +1 CN T - H +1 i n .0+3, T - H +1 CN o T - H o CN T - H o d T - H o CN N O T - H N O i n i n T - H N O .7+1. T - H +1 © 4+10 .4+4. i n +1 N O +1 O O NO oo' CN © CO m CN 0 0 CN > n CN o m N O C O NO c o 6+5. m +1 CN T - H +1 c o +1 +1 N O +1 q o d CN CN CN N O T—H i n T - H • n W T - H CN CO T t - i n C O 4-1 Bull 3 PQ 3 PQ 3 PQ 3 PQ Mean; 136 Figure 6.5. Differences in the percent binding of mAb I BS-1 to fresh and frozen-thawed spermatozoa of five bulls (mean+SE). 137 0 10 20 30 40 50 mAb binding (%) Figure 6.6. Correlation between mAb-binding and percent dead acrosome-intact spermatozoa (r=0.86; P<0.01). 138 Plate 6.4. Indiscriminate staining of bull spermatozoa by Hoechst 33342 at 0.1 tig/ml concentration. All spermatozoa in the field were stained, many live spermatozoa were seen moving with " fluorescent-heads". 139 Plate 6.5. Differential staining of bull spermatozoa by Hoechst 33258 at 0.5 jig/ml concentration. A) Spermatozoa photographed with both incandescent and fluorescent lamps on; note spermatozoa showing complete (dead; thick arrow), partial (live membrane-damaged; thick arrow-head) or no (live membrane-intact; thin arrow) staining. B) Same field photographed with only the fluorescent lamp on; note that unstained spermatozoa are not visible. 140 6.5. DISCUSSION Investigations on the biological applications of the newly generated mAbs conducted in this study have led to some interesting observations. None of the 13 mAbs tested had any inhibitory influence on sperm-zona binding, but one mAb (II BS-2) promoted sperm-zona binding to levels significantly higher than the control. We do not have a definite explanation for this observation at this time, pending further investigations. However, it is tempting to speculate that the cognate surface antigen of the mAb II BS-2 possibly plays an "anti-zona-binding" role, which, when inactivated by the binding of II BS-2, results in enhanced sperm-zona binding. If this is true, it would be somewhat similar to the finding of Leclerc et al. (1989; 1990) that heparin induced a decrease in the binding of calmodulin-binding proteins to calmodulin resulting in increased sperm capacitation and fertilization rates. Our findings suggest that the mAbs I BS-1,1 BS-5, I BS-12,1 BS-13, II BS-2 and II BS-11 are potential candidates for studying sperm surface changes associated with capacitation in vitro. Among the 8 mAbs of I BS series tested, only 4 showed an increase in binding to spermatozoa between 0.5 h and 4.5 h suggesting that the cognate antigens of these mAbs become more accessible with the progression of time. Since sperm viability did not change (despite a slight drop in motility) during this 4 h period, we have ruled out the possibility that the observed increase in mAb-binding may be due to an increase in dead sperm. Time-dependent increase in the binding of anti-human and anti-mouse sperm mAbs to spermatozoa incubated under capacitation conditions, as observed in this study, have been previously reported (Okabe et al., 1987; Fann and Lee, 1992; Liu et al., 1992; Archibong et al., 1995). This may be due to an increase in the permeability of sperm membranes associated with capacitation changes allowing passage of mAbs to access their specific antigens. Based on the findings of Liu et al., (1992) and their own, in studies using the anti-human sperm mAb HS-63, Archibong et al., (1995) 141 suggested that a surface pool of PSA-63, the cognate antigen recognized by HS-63, appears in the acrosomal cap region during sperm capacitation and prior to acrosome-intact sperm binding to the zona, and conclude that if this premise is true, the appearance of PSA-63 should be diagnostic of the capacitation process. In contrast to the mAbs of I BS series, mAbs of the II BS series II BS-2 and II BS-11 showed a decrease in binding to spermatozoa in the presence of heparin. Similar observations on the loss of sperm surface antigens during in vitro capacitation of human spermatozoa detected by an anti-human sperm mAb has been reported (Wolf et al., 1983). The findings of the present study would suggest that under conditions of heparin-induced capacitation, the antigens recognized by the mAbs are either removed from the sperm surface or inactivated, possibly by heparin itself. Heparin is well known to displace proteins from cell surfaces (Casu, 1985) and has been implicated in the removal of sperm surface proteins (Parrish and First, 1991; Miller and Hunter, 1986) on a time-dependent ( = 4 h) manner which may be indicative of capacitation. Since it is established that heparin binds with high affinity to bull spermatozoa (Handrow et al., 1984; Miller and Ax, 1990) there is a strong likelihood that heparin is responsible for the rapid decrease seen in the binding of mAbs II BS-2 and II BS-11. With the exception of three, all the mAbs of I BS series were found to be reliable bio-markers for evaluating acrosome status in bull sperm. The usefulness of 3 anti-human sperm mAbs for assessing acrosome status was reported earlier (chapter 3). One important advantage that mAbs have, over lectins, for assessing acrosome status is that lectin based techniques cannot be used in the presence of glycoconjugates such as cervical mucus or zona pellucida, as lectins would bind to them obscuring the sperm labelling pattern (Cross et al., 1986). The mAbs identified as markers for acrosome status in this study, therefore, have significant advantages in that they can be used as tools for identifying bull sperm acrosome status, not only in routine 142 laboratory evaluations, but also as specialized research tools to examine the status of spermatozoa removed from the female reproductive tract, even if they are in close association with cervical mucus, oviductal cells, cumulus cells or zona pellucida. These mAbs may therefore offer some hope for researchers looking at sperm transport in the female reproductive tract and sperm-uterus and sperm-oviduct interactions under in vivo situations. Methods to assess membrane integrity of frozen-thawed bull spermatozoa would be of significant advantage to the bovine Al industry. Fluorescent staining (Valcarcel et al., 1994), electron microscopy (Krogenoes et al., 1994), flow cytometry (Thomas and Garner, 1994) and enzyme-loss (Buhr and Zhao, 1995) have been used recently as measures for assessing post-thaw membrane integrity in bull and ram spermatozoa. We have attempted to use mAbs specific to intra-acrosomal antigens as indicators of membrane integrity of bull spermatozoa in this study. Blach et al. (1988) previously reported the use of an acrosome-specific mAb to assess plasma membrane integrity in stallion spermatozoa, but apprently no attempt has been made so far, to explore the use of mAbs to assess cryodamage in bull spermatozoa. It was shown that the four tested mAbs of I BS series bind to a significantly higher percentage of spermatozoa in frozen-thawed than fresh semen samples. In chapter 5, it was demonstrated that the I BS series mAbs neither bind to live membrane intact spermatozoa nor to acrosome-reacted (both live and dead) spermatozoa. Therefore, the hypothesis at the initiation of this study, was, that the mAbs would bind to dead acrosome-intact spermatozoa (assuming that all dead spermatozoa would have lost their plasma-acrosomal membrane integrity), and to plasma-acrosomal membrane damaged (but acrosome-intact) live spermatozoa. If the dead acrosome-intact population could then be identified by dual staining and excluded, the true population of live cryodamaged spermatozoa could be obtained. 143 However, the results were contrary to our expectation, in that, the percent mAb-binding and the percent dead acrosome-intact spermatozoa were closely related. It was expected that the mAbs will bind to a population of spermatozoa, marginally exceeding the dead acrosome-intact spermatozoa. But, in most situations, the percentage of mAb-binding was slightly lower than the percentage of dead acrosome-non-reacted sperm. This suggests that some dead spermatozoa may have retained their membrane integrity, thus preventing the mAbs from entering the cell. Krogenoes et al. (1994), after an ultrastructural study on frozen-thawed spermatozoa of Norwegian cattle, found that more than 90% of spermatozoa had intact membranes. Blach et al. (1989) also found that the plasma-acrosomal membrane integrity of most stallion spermatozoa was not altered sufficiently, to allow the binding of the mAb they tested. Though only two samples were tested, the results of the Hoechst 33258 staining also seem to support the view that the mAbs bind only to dead acrosome-intact spermatozoa. The Hoechst 33258 being a nuclear stain, will bind to four categories of spermatozoa, namely, dead acrosome-reacted, dead acrosome-intact, live acrosome-reacted and live membrane-damaged (acrosome-intact) spermatozoa. The mAbs, on the other hand, will not bind to acrosome-reacted sperm. This should explain why approximately a 10% higher staining was seen with Hoechst 33258 in both the tested samples. Alternatively, the permeability of sperm plasma-acrosomal membranes may depend on the magnitude of membrane damage. Thus, it is likely that the membranes could not exclude Hoechst 33258 and Trypan blue dyes as effectively as it could exclude the mAbs. The observation that both Hoechst 33258 and Hoechst 33342 could stain 100% of sperm at higher concentrations, only strengthens this latter possibility. The findings of the cryodamage assessment study did not support the hypothesis that the mAbs could be used for assessing cryodamage. Still, at this point of time, as the evidence we have presented is not conclusive, the possibility of using the mAbs for this purpose cannot be 144 totally ruled out. More detailed investigations in this direction, using electron microscopy, and mAb-labelling of spermatozoa in conjunction with a vital staining technique or direct immunofluorescence assays by directly conjugating the mAbs with FITC and examining wet smears of live-stained spermatozoa should offer a clear picture. In preliminary trials, the II BS series mAbs II BS-2 and II BS-11 were also tested as possible markers for assessing cryodamage. They generally showed a decreased binding to frozen-thawed than fresh spermatozoa. Since consistent results were not obtained, they are not presented. 6.6. CONCLUSION Some of the newly generated anti-bull sperm mAbs are promising candidates for investigating bull sperm surface changes associated with capacitation and acrosome reaction in vitro. Since they are highly specific to spermatozoa, and due to the non-species specific nature of some of the mAbs, there is a great potential for using these mAbs to study sperm surface changes in other mammalian species as well. Further investigations are needed to determine if the mAbs would be useful to detect cryodamage in frozen-thawed bull spermatozoa. 145 CHAPTER 7 FINAL DISCUSSION AND FUTURE CONSIDERATIONS 7.1. SUMMARY OF FINDINGS The aim of this thesis project was to evaluate anti-sperm monoclonal antibodies (mAbs) as markers for the in vitro assessment of surface changes associated with capacitation, acrosome reaction and cryopreservation in bull spermatozoa, and to examine the possible relationship between mAb-binding to spermatozoa and fertility. In the first set of experiments (chapter 3), three anti-human sperm mAbs crossreacting with bull sperm antigens were identified. All three mAbs exhibited a time-dependent increase in their binding to bull spermatozoa incubated under capacitation conditions. Binding was minimal initially, but reached maximum at 4 h of incubation, suggesting that either sperm membranes become more permeable to mAbs or the intra-acrosomal antigens migrate to become exposed during capacitation. Upon inducing acrosome reaction, the mAbs lost reactivity to spermatozoa, apparently due to the loss of their cognate antigens. Thus, the anti-human sperm mAbs HS-9, HS-11 and HS-63 appear to be potential markers for assessing sperm capacitation and acrosome status of bull spermatozoa under in vitro conditions. One mAb (HS-11) was chosen for further investigations. The bull sperm antigen recognized by HS-11 was purified by immunoaffinity chromatography and designated as BSA-11. The antigen consisted of two major proteins in the 18 to 20 kDa range. The immunological relatedness of BSA-11, its corresponding human sperm antigen HSA-11 and HSA-63 the cognate antigen of another anti-human sperm mAb HS-63, was demonstrated by ELISA. In spite of their 146 molecular size heterogeneity, the sperm antigen isolated from two different mammalian species were commonly recognized by all three mAbs. In chapter 4, the possible relationship between fertility and the binding of the anti-human sperm mAb HS-11 to bull spermatozoa was examined in a series of in vitro fertilization experiments. Semen samples of eight bulls were tested in four replicate trials. Distinct differences were seen in the binding of mAbs (22.0+8.3% to 51.8+5.2%, mean+SE) to sperm samples of different bulls. Results suggested a definite relationship between mAb-binding to spermatozoa at 4 h under capacitation conditions and fertility based on the ability of spermatozoa to induce cleavage of bovine oocytes in vitro. Yet, no relationship was found between HS-11 binding to spermatozoa and in vivo fertility based on 90-day non-return rate to first Al. With the hope of finding more suitable bio-markers, the raising of mAbs specific to bull sperm antigens was examined in the next study (chapter 5). After two successful cell fusion experiments, a total of 15 mAbs were generated, 11 specific to intra-acrosomal antigens and two specific to surface antigens of bull spermatozoa. Characterization studies revealed that all the mAbs were tissue-specific (specific to sperm) but not species-specific. Several of the mAbs crossreacted with spermatozoa of domestic livestock species (sheep, pig) while few crossreacted with spermatozoa of distantly related species (man, mouse). None of the mAbs crossreacted with rooster spermatozoa. The cognate antigens recognized by most of the mAbs were of testicular origin. However, the antigens apparently undergo post-testicular modifications when spermatozoa traverse the epididymis. Western blot analysis revealed that most of the antigens were of high molecular size (>200 kDa). The existence of at least five distinct antigenic domains in bull spermatozoa was demonstrated using these mAbs. The functional characterization of the newly generated mAbs was performed in the next series of experiments (chapter 6). To determine if the antigens recognized by the anti-bull sperm 147 mAbs play a role in fertilization, the influence of the mAbs on sperm-oocyte interaction was examined. Twelve of the thirteen mAbs tested had no effect on sperm-zona binding, while one surface-reacting mAb, II BS-2, enhanced the binding of spermatozoa to zona pellucida, suggesting that the antigen recognized by II BS-2 may have an "anti-zona-binding" role. As observed previously in chapter 3 with three anti-human sperm mAbs, four newly generated anti-bull sperm mAbs showed increased binding to spermatozoa incubated under capacitation conditions at 4 h, than at 0 h. Eight mAbs were tested and found to be useful markers for the determination of sperm acrosome status. In the last experiment of chapter 6, the possibility of using the mAbs as markers for assessing cryodamage in spermatozoa was examined. Four selected mAbs showed significant differences in their binding to fresh and frozen-thawed spermatozoa of the same ejaculate. Further studies revealed that the mAbs tend to recognize dead acrosome-intact spermatozoa but not live membrane-damaged spermatozoa. Therefore, the hypothesis that the mAbs would prove useful for assessing membrane damaged spermatozoa was not supported in this study. In summary, this project has demonstrated that anti-sperm mAbs could be used as bio-markers for the identification of bull sperm surface changes associated with capacitation and acrosome reaction in vitro. The correlation between the binding of the anti-human sperm mAb HS-11 to bull spermatozoa and in vitro fertility, suggests that the bull sperm antigen BSA-11 becomes exposed during capacitation allowing HS-11 to access and react with it. Further studies are needed to determine if the time-dependent changes in the reactivity of anti-bull sperm mAbs to bull spermatozoa is also related to fertility. 148 7.2. OVERALL DISCUSSION Anti-sperm mAbs have proved useful in enhancing the understanding of some of the mechanisms associated with sperm capacitation and fertilization, largely in laboratory animals, and to a lesser extent in the human. However, few studies have used anti-sperm mAbs in the evaluation of bull sperm changes in vitro. Artificial insemination (Al) for breeding has been maximally used in cattle, and perhaps there is no other species that has received so much attention in terms of semen evaluation. The objective of research on semen evaluation has been to predict fertility based on results of laboratory tests (Saacke, 1982; den Daas, 1992). In the past decades numerous tests have been developed for assessing the quality of semen used for Al. Even though enormous efforts have gone into research in this area, it has been impossible to develop a test that could predict fertility with certainty. The present study examined a non-conventional method of using anti-sperm mAbs as markers to evaluate bull sperm surface changes in vitro and their possible implications in fertility prediction. One of the major findings of this project was that certain anti-sperm mAbs specific to intra-acrosomal antigens show a time-dependent binding to bull spermatozoa incubated under capacitation conditions, with maximum binding occurring at 4 h of incubation. Previous studies have shown that bull spermatozoa need about 4 h to "capacitate" under in vitro conditions (Parrish et al., 1988; 1989; Fraser et al., 1995). It is also known that only capacitated spermatozoa would undergo acrosome reaction (Parrish et al., 1988; Yanagimachi, 1988) when induced to acrosome-react. Since it was found in this study that the percentage of spermatozoa undergoing acrosome reaction in response to lysophosphatidylcholine (LC) treatment and the percent spermatozoa reactive to mAb were closely related, it is suggested that the binding of mAbs to their cognate antigens in live spermatozoa is an indication of capacitation changes. 149 The present study also found that in addition to the three anti-human sperm mAbs crossreactive to bull spermatozoa, at least eight of the 15 newly generated anti-bull sperm mAbs are useful markers for the assessment of bull sperm acrosome status in vitro. Even though fluoresceinated lectins are powerful markers to detect acrosome status (Cross et al., 1986; Cross and Watson, 1994), since they tend to bind indiscriminately to glycoconjugates, lectins may not be effective for examining the acrosome status of spermatozoa closely associated with zona pellucida, cervical mucus or other female reproductive tract secretions. Reports indicate that bovine (Krogenoes et al., 1994) and human (Pijnenborg et al., 1985; Pereda and Coppo, 1987) cumulus cells exhibit pronounced phagocytosis of both intact and acrosome-reacted spermatozoa. However, little is known about the significance of this phenomenon. It is likely that molecular probes such as the mAbs raised in the present study will be useful in such investigations, and will definitely have an advantage over lectins for studies looking at in vivo sperm transport in cattle. The usefulness of anti-bull sperm mAbs specific to intra-acrosomal antigens as bio-markers to assess membrane-integrity/cryodamage in frozen-thawed bull spermatozoa could not be established with certainty. It was hypothesized that frozen-thawed spermatozoa would suffer substantial plasma-acrosomal membrane damage (resulting in a sizeable population of live membrane-integrity-compromised spermatozoa), and that the mAbs would readily bind to such membrane-compromised live spermatozoa, in addition to the dead acrosome-intact spermatozoa. Based on this hypothesis, it was expected that the mAb-bound spermatozoa would outnumber the dead spermatozoa, thereby allowing a calculation of the true membrane-damaged live sperm population. But the results obtained did not support our hypothesis. Staining of spermatozoa with supravital dyes and fluorescent labelled mAb(s) for simultaneous observation of sperm viability and mAb-binding in live spermatozoa may provide some definite answers. 150 Based on studies in chapter 4, it was found that the binding of the anti-human sperm mAb HS-11 to bull spermatozoa is correlated with in vitro fertility. However, no correlation was found between mAb-binding and in vivo fertility. Whether molecular changes of capacitation in vitro closely mimic those in vivo is still an open question (Bedford, 1983; Chavarria et al., 1992). In view of this, it is not known whether it is reasonable to expect a direct relationship between mAb-binding and in vivo fertility. Since the newly generated anti-bull sperm mAbs were not tested for the possible relationship between their binding to spermatozoa and in vitro/in vivo fertility, this is a difficult question to answer at this point of time, and would therefore need to be investigated furtner. One interesting observation in the present study was that the anti-bull sperm mAb II BS-2 significantly increased the binding of bull spermatozoa to homologous zona pellucida when incubated under conditions that favour capacitation. The specific role of the antigen recognized by II BS-2 in sperm-oocyte interaction and fertilization is not known at the present time. The existence of specific oviductal proteins that suppress sperm capacitation until shortly before ovulation has been suggested by Hunter (1986). Considering this, there is reason to speculate that similar to the oviductal proteins, the cognate sperm antigen of II BS-2 may be playing either an "anti-zona-binding" or an "anti-capacitation" role functioning as a "gate-keeper-protein" to prevent premature sperm capacitation. Such a mechanism may be essential to ensure successful fertilization. Even though millions of spermatozoa are placed in the female reproductive tract during mating/Al., only extremely few spermatozoa reach the site of fertilization (Cohen, 1975; Hunter etal., 1991; Drobnis and Overstreet, 1992; Hunter, 1993). Recent studies have provided evidence to show that the viability, motility and longevity of spermatozoa are enhanced in the presence of oviductal secretions or oviductal epithelial cells (Pollard et al., 1991; Ijaz et al., 1994). A synchronous development of the functionally capacitated state at a fixed moment in 151 essentially all spermatozoa inseminated might limit fertilization success (Bedford, 1983). The sequestration and delayed release of spermatozoa in oviductal reservoirs, therefore, may be nature's way of ensuring a supply of highly motile capacitated spermatozoa even hours after mating/Al, thus enhancing the chances for successful fertilization. In this context, it would be interesting to investigate the fate of the antigen recognized by II BS-2 in spermatozoa present in the oviductal mileu, and undergoing "delayed capacitation". Studies have shown the heterogeneous nature of the sperm population within an ejaculate (Cohen, 1975; Bedford, 1983; Fournier-Delpech and Thibault, 1993). The heterogeneous nature of bull spermatozoa within each ejaculate was obvious in this study also, since the mAbs bound only to a limited number of spermatozoa at any given time, when incubated live. Very high levels of binding were seen only when sperm-membranes were ruptured by methanol-permeation. It has been proposed that "fertilizing-sperm" are actively selected by the female immune system through immunoglobulins secreted into the female reproductive tract lumen in association with the leucocytic reaction (Cohen and Werret, 1975; Cohen and Tyler, 1980). Cohen and colleagues suggest that these antibodies bind only to a subpopulation of spermatozoa, which are eventually removed from the female reproductive tract, whereas spermatozoa not bound to antibodies become the "fertilizing-sperm" and are stored in the oviductal isthmus. They found that antibody-bound-spermatozoa decreased in frequency along the tract, with very few such spermatozoa present in the oviduct. Even though some evidence has been presented (Cohen, 1983; Drobnis and Overstreet, 1992) for the existence of selection within spermatozoa, the numerous complexities of the in vivo system, and the absence of molecular probes to assess the differences in sperm populations, have made it difficult to confirm this. The mAbs developed during the course of this thesis project may have some application in verifying the sperm-selection theory. 152 The existence of up to five major antigenic domains and a few more sub-domains in the bull spermatozoon has been demonstrated in this study for the first time. This, and the finding that several of the antigens are commonly shared by domestic livestock species offers hope for using these mAbs as tools for the isolation of specific sperm antigens (that may have functional significance) in a highly purified form. Some of the mAbs, such as I BS-3 and n BS-3, crossreacting with a wide spectrum of mammalian spermatozoa may help in the identification of different classes of animals on the evolutionary scale having common sperm antigen(s). 7.3. FUTURE CONSIDERATIONS Since it is apparent that anti-human and anti-bull sperm mAbs offer several exciting possibilities for a better understanding of sperm capacitation, acrosome reaction and sperm-oocyte interaction, detailed investigations should be conducted using the molecular probes that have become available as a result of this study. Some of the directions for future research are briefly mentioned here. An important question of immediate relevence to this project is to examine whether the reactivity of the newly generated anti-bull sperm mAbs to bull spermatozoa under capacitation conditions, is related to fertility. Even though the binding of the anti-human sperm mAb HS-11 to bull spermatozoa was correlated with in vitro fertility, no such relationship could be established between HS-11 binding and in vivo fertility. If a laboratory-based sperm fertility test of high predictive value could be developed, it would be of great advantage to the bovine Al industry. The mAbs I BS-1 and I BS-5 should therefore be examined for this purpose, due to the maximum increment in their binding to spermatozoa under conditions favouring capacitation. The decrease in the binding of the mAbs II BS-2 and II BS-11 to bull spermatozoa under the same conditions must also be examined for its relevance to capacitation and fertility. In order 153 to further define the relevance of mAb-reactivity to spermatozoa and capacitation, attempts should be made to answer the following questions: a) Do mAb-reactive live spermatozoa manifest hyperactivity? b) Are only the "antigenically-previleged" spermatozoa reacting with the mAb(s), capable of binding to zona-pellucida? c) Is it possible to segregate mAb-reactive and mAb-non-reactive spermatozoa by flow-cytometry? If so, would such flow-cy to metrically sorted "enriched population of capacitated spermatozoa" yield better results in a bovine IVF system. The observation that II BS-2 consistently enhances sperm-zona binding under in vitro conditions is another aspect which needs to be investigated in detail. Even though sperm-zona binding is enhanced by II BS-2, it is not known if a similar effect would be seen on sperm penetration. Bovine IVF experiments designed to test the effect of the mAb II BS-2 on sperm penetration and fertilization must hence be initiated. It would be worth investigating if the rate of fertilization could be increased by co-incubating spermatozoa with II BS-2. Particular attention should be paid to the incidence of polyspermy in fertilized oocytes. The applications of the anti-sperm mAbs in the assessment of cryodamage in bull spermatozoa must be re-investigated as the results in this study have not been conclusive. The dramatic phenomenon of the transfer of sperm surface proteins to the egg during fertilization, and their persistance in the preimplantation embryo has been recorded in the mouse, rat and sea urchin (Gabel et al., 1979; Guant, 1983; Gunderson and Shapiro, 1984). It is quite likely that similar mechanisms exist even in large mammalian species. 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Expl Cell Res 89:161-174. 175 APPENDIX A COMPOSITION OF MODIFIED TYRODE'S MEDIA, SP-TALP AND FERT-TALP Unit Sp-TALP H-TALP Fert-TALP Nacl mM 100.0 100.0 114.0 KC1 mM 3.1 3.1 3.2 NaHCOj mM 25.0 25.0 25.0 NaH2P04 mM 0.3 0.3 0.3 Sodium lactate mM 21.6 21.6 10.0 Cacl2 mM 2.0 2.0 2.0 MgCl2 mM 0.4 0.4 0.5 HEPES mM 10.0 10.0 -Sodium pyruvate mM 1.0 1.0 0.2 BSA mg/ml 6.0 6.0F 6.0F Gentamicin Mg/ml 50.0 50.0 50.0 Heparin sulfate itg/ml - 10.0 10.0 F = fatty acid free 176 CO 0 > > O * o N C D s > E O 2 - Q > o PQ Q O W H CO 0 5 N 0) O i/ml -4—' 2 _ c c o a pm t » S: o O T _ pm "3 o elo tro x > CD c ° ° n O ° - o o . oo o PQ COO o ° o r> oo - o > ° o CD O O) CO > CO 0 O CO a> CO > O Q-_J £ Li. > o V CO LL CO CO L L E Ha o o CO o E CD co 0 CO c CO 177 APPENDIX C DUAL STAINING TECHNIQUE FOR VIABILITY AND ACROSOMAL STATUS ASSESSMENT Stain preparation TRYPAN BLUE (0.2% solution) Dissolve 200 mg Trypan Blue dye in 100 ml Sp-TALP medium (prepared without adding BSA) and filter. GIEMSA STOCK Dissolve 1.0 g Giemsa dye in 54 ml glycerol (warming may be necessary). Cool and add 84 ml methanol. Stir well and filter. Staining procedure 1. Mix 100 ill Trypan blue (0.2% solution) with 100 /d of medium containing spermatozoa. 2. Incubate on watch glass at 37-38°C for 3 minutes. 3. Prepare thin smears on clean glass slides and dry for 15 minutes at room temperature. 4. Dilute the Giemsa stock (0.72%) three times with distilled water and make a working solution. 5. Stain the previously Trypan blue stained smear by immersing in freshly diluted Giemsa working solution for 1-2 hours at room temperature. 6. Dry the smears between folds of clean filter paper, but do not rub. Remember that there is no washing involved at any stage. 7. Examine smears under oil immersion without coverglass. 8. Count at least 200 spermatozoa from each sample and classify as: a) live sperm with intact-acrosome, b) dead sperm with intact acrosome, c) live sperm without acrosome, and d) dead sperm without acrosome. (Procedure modified from Sidhu et al., Biotechnic Histochem. 1992: 67:35-39) 178 APPENDIX D CELL FUSION PROTOCOL Solution A HAT (50x) Lipopolysaccharide Thymocyte IMDM (lx), to make Fetal calf serum 10 ml 2 ml 0.2 ml (final cone. 133 /xg/ml) 2 x 10' 15 ml Fusion Mix NS-1 myeloma cells (2 x 107 in 5 ml IMDM) and Spleen cells (1 x 108 in 5 ml IMDM) in 1:5 ratio. Spin at 1000 rpm for 5 minutes Add 0.7 ml 50% PEG in 1 minute @ 0.1 ml each addition Sitr slowly and thoroughly for 1 minutes Add 2 ml IMDM (serum free), mix slowly and thoroughly in 2 minutes Add 7 ml IMDM (serum free), mix slowly and thoroughly in 2 minutes Spin at 1000 rpm for 5 minutes Add 15 ml solution A and mix slowly Mix well with 25 ml 2% methylcellulose in IMDM Pour into 35 mm dishes @ 1.5 ml per dish COMPOSITION OF BUFFERS FOR CELL FUSION 2X IMDM Powder medium (1 pkt) 17.70 g NaHC03 3.024 g Alpha-thioglyecrol 6.0 /d Pencillin-Streptomycin 100X 10 ml Make up to 500 ml in Milli-Q-water 179 2% Methylcellulose 2 g methylcellulose in 50 ml distilled water autoclaved for 15 minutes at 15 psi (compensate weight loss during autoclaving with distilled water) 50% Polyethylene glycol 2 g PEG autoclaved for 15 min at 15 psi. Add 2 ml IMDM when the PEG solution is still warm. RPMI complete medium RPMI lOx d H 20 FCS (heat inactivated) Penicillin-Streptomycin HT (50x) NaHC03 (7.5%) Adjust pH to 7.0 with 1 N NaOH Glutamine (lOOx) RPMI serum-free medium RPMI lOx 48.5 ml d H20 440 ml NaHC03 11 ml NaOH 1.5 ml Penicillin-Streptomycin 5.0 ml 45 ml 375 ml 50 ml 10 ml 10 ml 11 ml 5 ml 180 APPENDIX E PROTOCOL FOR ASSESSING SPERM ANTIGEN LOCALIZATION BY SCANNING ELECTRON MICROSCOPY 1. Wash spermatozoa in PBS (pH 7.4) twice, by centrifugation at 250 x g for 3 minutes each time. 2. Incubate with primary antibody (hybridoma culture medium 1:1 or ascites fluid 1:100) for 30 minutes (39°C) in PBS + 1 % serum at pH 7.4 [FOR SURFACE ANTIGENS] 3. Wash 3 times in PBS. 4. Incubate with goat antimouse IgG+IgM conjugated with 10 nm gold particles (30-40 nm size particles would be preferred, if available) diluted 1:50 with PBS @ pH 7.4 containing 0.5% BSA for 30 minutes [Optional step: At this stage, aliquots of sperm suspension may be spotted to slides, counter-stained with silver enhancer stain to monitor the presence of immunogold label by light microscopy before proceeding to stages required for electron microscopy] 5. Wash 3 times in PBS. 6. Fix in 2.5% glutaraldehyde in 0.1 M Sodium cacodylate buffer of pH 7.4 for 30 minutes. 7. Wash 3 times with 0.1 M Sod. cacodylate buffer pH 7.4 (10 min each wash) 8. Osmicate (1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4) for 60 minutes at room temperature The specimen could then be transferred to an inverted 20 ml syringe fitted with a disc filter (pore size l^ m) for convenience of the washing and dehydration steps. 9. Wash 3 times with distilled water (10 minutes each) 10. Dehydrate in ascending grades of ethanol (70%, 85%, 95% each once) and (100% twice), giving 10 minutes at step. 11. Remove the disc filter and run through Critical Point Dryer (CPD) for 1 h 20 min. 12. Cut the filter, mount on stubs and gold coat for short time (2-3 minutes). 181 APPENDIX F PROTOCOL FOR SPERM-ZONA BINDING ASSAY 1. Collect oocytes from frozen-thawed ovaries either by slicing or aspiration, in Dulbecco's phosphate buffered saline supplemented with 0.4% BSA. 2. Thaw 1-2 straws of semen and wash in Sp-TALP medium three times. 3. Concentrate and resuspend in H-TALP or Fert-TALP medium. 4. Adjust volume to attain a sperm concentration of 10xl06/ml. 5. Draw 50 fA and place in microtitre wells. Prepare wells sufficient for available oocytes (@ 10 oocytes/well). 6. Add 50 fA of the hybridoma culture supernatant to the pre-labelled well (now the sperm cone, will be 5xl06/ml). Add fresh culture medium to control well. Incubate for 30 minutes. 7. Using fine bore pipette, remove cumulus cells from oocytes and add @ 10 oocytes per well, in 10 /xl medium. DO NOT SELECT oocytes. 8. Coincubate sperm, mAb and oocytes for 4 hours. 9. Pick and move oocytes to next well. Remove loosely attached sperm by pipetting up and down TEN TIMES (you may vary this, but be consistent). 10. Put 2.5% glutaraldehyde in one row of wells (25-50 /xl/well). 11. Transfer eggs to glutaraldehyde and allow a fixation time of 10 minutes. 12. Wash each group of oocytes three times in PBS (drops made in 35 mm dishes will be convenient for washing). 13. Stain with Hoechst 33342 stain for 10 minutes (1 mg/ml) in dark chamber. Wash again with PBS three times. 14. Transfer the sperm-oocyte complexes to glass slides, slightly compress under a coverslip supported on the corners by vaseline or wax, and seal with nail polish. 15. Keep in a dark, cool place until ready to read in epifluorescence microscope. 16. Use UV-2A filter (excitation:330-380 nm; barrier:420 nm). 182 

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