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The effects of progesterone and cholesterol on sperm capacitation, acrosome reaction, and in vitro embryo… Motamed Khorasani, Afsaneh 1999

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T H E E F F E C T S O F P R O G E S T E R O N E AND C H O L E S T E R O L ON S P E R M C A P A C I T A T I O N , A C R O S O M E R E A C T I O N , AND IN VITRO E M B R Y O PRODUCTION IN C A T T L E by A F S A N E H M O T A M E D KHORASANI B.Sc, The Medical University of Shahid Beheshti, Tehran, Iran, 1992 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R S O F SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Animal Science) We accept this thesis as conforming to the requiredjstan£a,rd THE UNIVERSrf Y O F BRITISH COLUMBIA April 1999 Afsaneh Motamed Khorasani, 1999 U B C Spec ia l Co l lec t ions - Thes i s Author isat ion Fo rm P a g e 1 of 1 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of /^/i/ynaJ l y w ' ^ C A The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date dpr// /qqf http://www.library.ubc.ca/spcoll/thesauth.html 6/30/99 A B S T R A C T Progesterone (P4) is known to induce mammalian sperm acrosome reaction (AR) in vitro, whereas cholesterol (CH) one o f the major components in seminal plasma, inhibits A R . The objectives o f this study were: 1) to elucidate the in vitro effects of P4, 17 P-estradiol (E 2 ) , C H , and their combination on cryopreserved bovine sperm A R , 2) to study the mechanisms o f C H action on P4-induced A R , and 3) to determine the effects o f exogenous P4 and C H on zona-binding ability o f bovine sperm and in vitro fertilization (IVF). For the first two objectives, swim-up separated sperm from six bulls were incubated in modified Tyrodes* medium for 0-6 h (38.5°C, 5% CO2). Acrosome reaction was determined following addition o f P4 (10 p.g/m.1), E2 (10 ug/ml), d b - c A M P (1 mM) , forskolin (10 uM) , and Ca 2 +- ionophore (10 pJVI), either in the presence or absence of C H (1 (tg/ml). The effects of P-sitosterol (1 p.g/ml), a C H plant analog, and C H on P4-induced A R were also compared. For zona-binding assay, thawed sperm were washed in modified Tyrodes' medium. Sperm (0.2 x 10 6 sperm/ml) were pre-treated with P4 (10 pig/ml), either in the presence or absence o f C H (1 }t.g/mi) at 38.5°C, 5% CO2. Sperm were co-incubated with cumulus-free oocytes for 18 nr. Fol lowing washing, f ixing, and staining, the number o f sperm attached to oocytes was counted under a fluorescent microscope. For IVF , 30 min prior to the addition of matured oocytes, sperm (0.2 x 10 6 sperm/ml) were pre-treated with P4 (10 |^g/ml), C H (1 jtg/ml), combination of P4 (10 ug/ml) and C H (1 p.g/ml), and cyclodextrin (10 p.g/ml). Sperm exposed oocytes were then incubated in modified Tyrodes' medium (supplemented with 10% serum) at 38.5°C and 5% CO2. Cleavage rates, as an indicator o f fertilization, were recorded after 72 nr. Acrosome reaction was induced by P4 (34.4 ± 2.5%), E 2 (4.5 ± 0.7%), db - cAMP (49.3 ± 2.0%), forskolin (81.8 ± 1.2%), and Ca 2 +- ionophore (95.7 ± 1.0%) immediately after-thawing (p < 0.05 for all). Cholesterol and P-sitosterol inhibited P4-ii induced A R in an identical manner (95.6% and 95.4% inhibition, respectively). Dibutyryl-cAMP- induced A R was also inhibited by C H but this was not the case for forskolin and Ca -ionophore. Progesterone increased both the number o f spermatozoa bound to zona (73.9 ± 3 . 7 compared to 45.8 ± 2.8 for control, 46.2 ± 3.3 for C H , and 45.3 ± 1.8 for P 4 + C H ) and the percentage of cleavage rate (42.5 ± 3.8% compared to 22.5 ± 2.1% for control, 18.3 + 2.8% for C H , 20.8 ± 2.0% for p-cyclodextrin, and 17.5 ± 1.7% for P 4 + CH) . From this study it is concluded that a) C H inhibits A R induced by P 4 as wel l as by d b - c A M P , b) cholesterol inhibits A R possibly by modifying the structure o f the sperm plasma membrane, and c) progesterone treatment of sperm immediately prior to fertilization increases cleavage rate and may improve mammalian IVF success rate. iii T A B L E O F C O N T E N T S A B S T R A C T 11 L IST OF T A B L E S vi LIST OF FIGURES Vll L IST OF P L A T E S ix A B B R E V I A T I O N S x A C K N O W L E D G E M E N T S Xll F O R E W O R D xiii C H A P T E R 1. G E N E R A L I N T R O D U C T I O N C H A P T E R 2. A C O N C E P T U A L O V E R V I E W 5 2.1. S T R U C T U R E O F T H E M A M M A L I A N S P E R M A T O Z O A 2.2. S P E R M C A P A C I T A T I O N 2.3. S P E R M A C R O S O M E R E A C T I O N 2.4. S T R U C T U R E OF T H E M A M M A L I A N O O C Y T E 2.5. F E R T I L I Z A T I O N 2.6. L A B O R A T O R Y P R O C E D U R E S F O R F E R T I L I T Y E V A L U A T I O N 2.7. IN VITRO F E R T I L I Z A T I O N IN C A T T L E C H A P T E R 3. E F F E C T S OF 17 p - E S T R A D I O L , P R O G E S T E R O N E , A N D C H O L E S T E R O L O N S P E R M A C R O S O M E R E A C T I O N A N D M E C H A N I S M O F C H O L E S T E R O L E F F E C T S 39 3.1. I N T R O D U C T I O N 3.2. M A T E R I A L S A N D M E T H O D S 3.3. R E S U L T S 3.4. D I S C U S S I O N S 3.5. C O N C L U S I O N S C H A P T E R 4. E F F E C T S OF P R O G E S T E R O N E A N D C H O L E S T E R O L O N S P E R M A N D O O C Y T E I N T E R A C T I O N IN IN VITRO F E R T I L I Z A T I O N S Y S T E M 84 4.1. I N T R O D U C T I O N 4.2. M A T E R I A L S A N D M E T H O D S 4.3. R E S U L T S 4.4. D I S C U S S I O N S 4.5. C O N C L U S I O N S C H A P T E R 5. F I N A L D I S C U S S I O N A N D F U T U R E POSSIBIL IT IES 114 5.1. S U M M A R Y OF F INDINGS 5.2. O V E R A L L D I S C U S S I O N 5.3. F U T U R E POSSIBIL IT IES R E F E R E N C E S 121 A P P E N D I X A . C O M P O S I T I O N OF I V F M E D I A (BO M E D I U M ) 151 A P P E N D I X B. C O M P O S I T I O N OF M O D I F I E D T Y R O D E ' S M E D I A ( S P - T A L P , H - T A L P , A N D F E R T - T A L P ) 154 A P P E N D I X C. S T E P B Y STEP B O V I N E I V F P R O C E D U R E S ( T A L P M E D I U M ) 155 A P P E N D I X D. I L L U S T R A T E D B O V I N E IVF P R O C E D U R E S (BO M E D I U M ) 156 A P P E N D I X E. C H E M I C A L S T R U C T U R E S (17 P -ESTRADIOL, P R O G E S T E R O N E , C H O L E S T E R O L , p - C Y C L O D E X T R I N , p -S ITOSTEROL) 157 A P P E N D I X F. P R O T O C O L F O R S P E R M P S A - F I T C S T A I N I N G D I R E C T I M M U N O F L U O R E S C E N T A S S A Y 159 A P P E N D I X G . P R O T O C O L F O R S P E R M - Z O N A B I N D I N G A S S A Y 160 A P P E N D I X H. P R E L I M I N A R Y STUDIES 161 LIST O F T A B L E S T A B L E 3.1. Treatments carried out in Chapter 3 51 T A B L E 3.2. The average percentages of acrosome-intact sperm from all bulls at 0, 2, 4, and 6 hours o f incubation in capacitation medium (n = 72) 57 T A B L E 3.3. The average percentages of acrosome intact sperm in six individual bulls at 0, 2, 4, and 6 hours of incubation in capacitation medium (n = 72) 58 T A B L E 3.4. The average percentages of sperm motility/viability decrease in all bulls during 6 hours o f incubation in capacitation medium (n = 18) 61 T A B L E 3.5. The average percentages of sperm viability decrease in six bulls during six hours o f incubation in capacitation medium (n = 18) 63 T A B L E 4.1. Effects of progesterone and cholesterol on cleavage and blastocyst formation rates using B O medium (sperm concentration of 5 x 10 6 sperm/ml, n = 3) 101 T A B L E H . l . The percentages of A R induced by different concentrations of 17 P-estradiol (in 18 replicate experiments) 161 vi LIST O F FIGURES F I G U R E 3.1. The percentages of progesterone-induced A R in six bulls (n = 72) 59 F I G U R E 3.2. The percentages of sperm motility decrease in six bulls during 2, 4, and 6 hours of incubation in capacitation medium (n = 18) 62 F I G U R E 3.3. The percentages of sperm motility and viability decrease in six bulls during 2 ,4 , and 6 hours o f incubation in capacitation medium (n = 18) 63 F I G U R E 3.4. The percentages of sperm A R increase following 17 p-estradiol treatment in six bulls (n = 72) 65 F I G U R E 3.5. The effects o f cholesterol and P-sitosterol on progesterone-induced A R of frozen-thawed bovine sperm (n = 72) 67 F I G U R E 3.6. The effects of cholesterol on progesterone-, db - cAMP- , forskolin, and calcium ionophore-induced A R of frozen-thawed bovine sperm (n = 72) 68 F I G U R E 3.7. The percentages of A R increase following db -cAMP treatment in six bulls (n = 72) 69 F I G U R E 3.8. The percentages of A R increase following forskolin treatment in six bulls (n = 72). . 70 F I G U R E 3.9. The percentages of A R increase following Ca 2 +- ionophore treatment in six bulls (n = 72) 73 F I G U R E 3.10. The percentages of A R increase following progesterone, 17 P-estradiol, db-c A M P , forskolin, and Ca 2 +- ionophore treatment in six bulls (n = 72) 74 F I G U R E 4.1. Effects of sperm concentration on the percentages of cleavage rate and blastocyst-formation in bovine IVF system (n = 3) 102 F I G U R E 4.2. Effects of progesterone and cholesterol on cleavage rates using B O medium (sperm concentration of 0.2 x 10 6 sperm/ml, n = 6) 104 F I G U R E 4.3. Effects of progesterone and cholesterol on cleavage rates using T A L P medium (sperm concentration o f 0.2 x 10 6 sperm/ml, n = 6) 105 F I G U R E 4.4. Effects o f progesterone and cholesterol on cleavage rates using T A L P medium (sperm concentration of 0.2 x 10 6 sperm/ml, n = 6) 106 F I G U R E 4.5. Effects of progesterone and cholesterol on number of zona-bound sperm (n = 3). 107 vii F I G U R E H . l . Percentages of A R induced by 30-minute incubation with different concentrations of progesterone (n = 18) 161 F I G U R E H.2. The percentages of sperm A R increase following addition of progesterone to different concentrations of cholesterol (n = 18) 162 viii LIST O F P L A T E S P L A T E 2.1. Schematic structure of mammalian spermatozoon 13 P L A T E 2.2. Schematic structure of mammalian oocyte after ovulation 30 P L A T E 3.1. Schematic illustration o f acrosome-intact and acrosome-react spermatozoa stained with F I T C - P S A 50 P L A T E 3.2. Progesterone-induced A R 60 P L A T E 3.3. Calc ium ionophore-induced A R 72 P L A T E 4.1. Freshly removed bovine oocyte from the ovary 91 P L A T E 4.2. Two-celled bovine embryo. 92 P L A T E 4.3. Four-celled bovine embryo 92 P L A T E 4.4. Eight-celled bovine embryo 93 P L A T E 4.5. Hatching bovine embryo 93 P L A T E 4.6. Hatched bovine embryo 94 P L A T E 4.7. Zona-binding assay with bovine sperm and oocyte 108 ix A B B R E V I A T I O N S A l = Art i f ic ial Insemination A N O V A = Analysis of Variance A R - Acrosome Reaction A T P = Adenosine Triphosphate B S A = Bovine Serum Albumin C = Carbon atom [Ca 2 + ] = Calc ium Ion Concentration c A M P = Adenosine 3':5'-Cyclic Monophosphate d b - c A M P = 2'-0-Dibutyryl Adenosine 3':5'-Cyclic Monophosphate D M S O = Dimethylsulfoxide D N A = Deoxyribonucleic Ac i d E C S = Estrus Cow Serum F B S = Fetal Bovine Serum F I T C = Fluorescein Isothiocyanate F I T C - P S A = Fluorescein Isothiocyanate-Labeled P S A F S H = Fol l ic le Stimulating Hormone g = Gravity gm = Gram h r = Hour I A M - Inner Acrosomal Membrane I M P = Intramembranous Particles I U = International Unit I V F = In Vitro Fertilization k D a = K i l o Dalton mg = Mi l l igram min = Minute mM = Mi l l imolar nm = Nanometer O A M = Outer Acrosomal Membrane p = Probability (p Value) P B S = Phosphate Buffered Saline X PH = -Log [H+] PSA = Pisum Sativum Agglutinin (a lectin) r = Co-Efficient of Regression R H = Relative Humidity SAS = Stat is t ical Analysis System SE = Standard Error sec = Second SOCS = Super Ovulated Cow Serum S P M = Sperm Plasma Membrane T A L P = Tyrode's Albumin Lactate Pyruvate ul = Microlitre u,m = Micrometer u.M = Micromolar = Microgram ng = Nanogram W H O = World Health Organization xi A C K N O W L E D G E M E N T S First and foremost, I wish to express my appreciation to Dr. R. Rajamahendran for being my thesis supervisor and for his guidance during the project. I should also thank Dr. G . Lee for being my co-supervisor, for his support and guidance during the project, and for allowing unrestricted access to his laboratory facilities. I am also thankful to Dr. K. M . Cheng for his cooperation and assistance as the graduate coordinator and the technical advice for statistics. I thank Dr. JR. Thompson for being the Chair of my final exam committee. I feel thankful to all these people for serving on my advising committee, and for offering their helpful suggestions. I appreciate the support o f all the faculty and staff members o f the Department o f An imal Science. I should mention Shehnaz, Donna, Gi l les, Si lv ia, and Siva for their assistance with office/laboratory procedures. I also feel thankful to Westgen, Mi lner, for supplying the semen samples for this study. I am grateful to The University o f Brit ish Columbia for awarding me a Graduate Student Bursary, the Faculty o f Graduate Studies for awarding me a travel grant, The Department o f Medical Genetics for awarding me a two-term Project Assistantship, and the Department of Obstetrics and Gynecology for awarding me the Graduate Research Assistantship for almost one year o f my study. The encouragement and prayers of my dear parents has surely had an essential role in the accomplishment of this work. I am greatly thankful to the Lord to whom I deeply believe and to whom I owe all accomplishments in my life. A t last, it would be almost impossible to complete this work without understanding and support o f my dear husband, Mehdi , whose patience is greatly admired. xii F O R E W O R D This thesis is based on the following manuscripts, which have either been published or soon to be submitted for publication: 1. C . Y . G . Lee, A . Motamed Khorasani, S. Dorjee (1998) Assessment o f progesterone-induced acrosome reaction by biotinylated monoclonal antibody probes. A m J Reprod Immunol 39:164-171. 2. A . Motamed Khorasani, A . P . Cheung, C . Y . G . Lee (1999) Mechanisms of cholesterol inhibitory effects on progesterone-induced acrosome reaction of human sperm. M o l Human Reprod (in press). 3. R. Rajamahendran, A . Motamed Khorasani, C . Y . G . Lee (1999) The effects of progesterone and cholesterol in acrosome reaction of bovine cryopreserved sperm, (accepted for presentation at the 3 2 n d Annual Meeting of Society for the Study of Reproduction, 1999; to be submitted for publication). 4. A . Motamed Khorasani, R. Rajamahendran, C . Y . G . Lee (1999) The effects o f progesterone and cholesterol in in vitro embryo production and zona-biding assay in cattle, (accepted for presentation at the 3 2 n d Annual Meeting of Society for the Study of Reproduction, 1999; to be submitted for publication). xiii C H A P T E R 1 G E N E R A L I N T R O D U C T I O N Just after leaving the testis, mammalian spermatozoa have neither progressive motility nor the ability to fertilize oocytes. During epididymal transit, sperm acquire the ability to move progressively but fertilization capacity is gained only after residence in the female reproductive tract for a finite period o f time. Two major changes occur in sperm prior to fertilization, an initial sperm membrane alteration (Yanagimachi, 1981; Langlais and Roberts, 1985) and the fusion of the plasma membrane and outer acrosomal membrane (Yanagimachi and Usu i , 1974). The first set o f changes which is generally termed capacitation, includes a series o f reversible changes allowing the influx o f calcium ions (Chang, 1951, 1955; Austin, 1952; Florman and Babcock, 1991; Yangimachi, 1994; Cross, 1998; Florman et al, 1998). The second set o f changes which is termed acrosome reaction (AR) , is one of the most critical sperm physiological functions during the initial fertilization process, both in vivo and in vitro (Yanagimachi, 1981,1988). There are many different inducers for A R . Zona pellucida can initiate the A R in vitro and is generally considered to be the in vivo inducer of A R (Cross and Morales, 1988; K o p f G S et al, 1991). However, mammalian sperm A R also can be initiated in vitro by suitable alternative agents like fusogenic lipids (Ohzu and Yanagimachi, 1982; llanos and Meize l , 1983), calcium ionophores (Russell, 1979; Ai tken et al., 1984; Tesarik, 1985; Yanagimachi, 1994; Brucker and Lipford, 1995), oocyte-cumulus complex (Siiteri, 1988; Stock et al, 1989), follicular f luid (Tesarik, 1985; Suarez et al, 1986; M c Atee and Dawson, 1989; Blumenfel and Nahlas, 1989), and progesterone (Osman et al, 1989; Thomas and Meize l , 1989; Meize l et al, l 1990; Blackmore et al, 1990; Melendrez et al, 1994; Roldan et al, 1994; Meyers et al, 1995; Leeet al, 1998). Progesterone is a steroid mainly secreted by the corpus luteum, but it is also secreted by cumulus oophorus-follicular cells that remains associated with the ovulated zona-enclosed oocyte. Since the cumulus is present at the time of fertilization, progesterone, secreted by the cumulus cells may have a role in initiating the A R of fertilizing sperm in vivo (Osman et al, 1989; Me ize l et al, 1990). Progesterone also appears to stimulate capacitation o f human and porcine sperm (Das Gupta et al, 1994; Barboni et al, 1995). However, the precise role o f progesterone on the signal transduction pathways involved in A R is still not wel l understood although interactions between progesterone and its receptors on the sperm surface have been implicated (Hyne and Garbers, 1979; White and Aitken, 1989; De Jonge et al, 1991; Rotem et al, 1992; Furuya et al, 1993; Bielfeld et al, 1994). Recent studies in human spermatozoa suggest that progesterone-induced A R is involved in both an early and a latent calcium influx by triggering the opening o f a plasma membrane calcium channel and activating protein-tyrosine kinase (PTK) , respectively (Tesarik et al, 1993a, Luconi et al, 1995). Different progesterone receptors might be involved in these two distinct reactions (Blackmore et al, 1991b; Mendoza et al, 1995; Sabeur et al, 1996; Tesarik et al, 1996). The first objective of this study is to elucidate the role o f steroids (progesterone, 17 0-estradiol, and cholesterol) on bovine sperm A R as wel l as to elucidate the capacitation status o f cryopreserved bovine sperm. The results o f the current study may be potentially useful for improving the environment of in vitro fertilization (IVF) so that a higher fertilization rate can be achieved. Insufficient A R in human sperm during the sperm-egg interaction, or under in vitro conditions, spontaneous or progesterone-induced A R , has been implicated cl inically to be associated with certain male factor infertility (Stock and Fraser, 1987; Tesarik and Mendoza, 2 1992). Moreover, progesterone-induced A R can be used as an alternative for acrosomal assessment of human sperm (Lee et al, 1998). If a significant correlation between progesterone-induced A R in bovine sperm and the in vivo fertility of semen can be demonstrated, a laboratory test may be developed to predict field fertility o f bulls. Another aspect o f this thesis is to further elucidate the effects o f cholesterol on the cryopreserved bovine sperm and its cellular mechanism of effects on mammalian sperm. Sperm plasma membrane has a unique l ipid composition, which is different from that of somatic cells (Poulos and White, 1973; Mack et al, 1986; Alvarez and Storey, 1995). Significant changes are known to occur in the plasma membrane during sperm maturation, as wel l as during capacitation in the female reproductive tract, in vivo. Among these l ipid components, cholesterol plays a major regulatory role on the fluidity of the l ipid bilayers such as membrane permeability, the lateral mobility of integral proteins, and functional receptors within the membrane (Langlais and Roberts, 1985; Benoff, 1993a). Cholesterol is also known to be one o f the major components of seminal plasma that inhibits human sperm A R (Davis, 1976a; Ehrenwald et al, 1988a, 1988b; Go and Wolf, 1985; Sugkraroek et al, 1991; Benoff et al, 1993b; Zarintash and Cross, 1996). Progesterone-induced A R is inhibited by the presence of cholesterol in human sperm (Zarintash and Cross, 1996; Lee et al, 1998). However, the underlying mechanisms involved, specially its inhibitory effect on progesterone-induced A R , remain unknown. Cholesterol may have a direct effect on the signal transduction pathways through a receptor-mediated process. Alternatively, the presence o f cholesterol may alter the structure or fluidity o f sperm plasma membrane, thereby exposing progesterone surface receptors for ligand binding and action. In this thesis, experiments were performed to further elucidate the cellular mechanisms o f cholesterol effects on progesterone-induced A R , in bul l spermatozoa. 3 Lastly based on the results of my previous studies in humans and also the present study in bovine sperm, progesterone was observed to induce sperm A R effectively. In contrast cholesterol inhibited the progesterone effects. Therefore, it may seem logical to assume the possibility o f utilizing these chemicals to manipulate the fertility both in vivo and in vitro. In order to examine this possibility animal models (bovine sperm and oocytes) were used because o f ethical problems involved in using human cells. The actual effects o f progesterone and cholesterol on the in vitro system of embryo production in cattle as wel l as their effects On sperm-zona binding have been elucidated in the present thesis. 4 CHAPTER 2 A CONCEPTUAL OVERVIEW The main objective of this chapter is, to provide a brief review of the structure and function of spermatozoa including its modification during epididymal maturation, capacitation, and AR. A brief review of the oocyte structure and the mechanism of fertilization has been also presented and discussed. Finally, the current laboratory procedures available for fertility evaluation and in vitro fertilization in cattle are reviewed. 2 . 1 . Structure of the mammalian spermatozoa Mature mammalian spermatozoa are elongated cells consisting of a flat oval head containing the nucleus and a tail (flagellum), containing the apparatus necessary for cell motility (Saacke and Almquist, 1964a, 1964b; Garner and Hafez, 1993). The overall length of a typical mature spermatozoon is between 68 to 74 urn. The head is 8 to 10 um long, 4 to 5 um wide, and 0.3 to 0.5 um thick (Sullivan, 1978). A short neck connects the sperm head and its tail, which is 0.3-1.5 urn. The tail is further divided into the middle (10 um long and 0.85 um in diameter), principal (45-50 um long), and end pieces (2-4 um long) (Garner and Hafez, 1993). 2 . 1 . 1 . The spermatozoa organelles The outermost structure of spermatozoa is the plasma membrane (plasmalemma). Underlying the plasma membrane is acrosome which is a double-layered structure located between the plasma membrane and the anterior portion of the nucleus. Several hydrolytic enzymes are stored within this cap-like structure involved in the fertilization process (Garner 5 and Hafez, 1993). The nucleus, which lies underneath the acrosome and is enclosed by a nuclear membrane, is known to contain the condensed chromosomes or genetic material (Garner and Hafez, 1993). The mitochondrial sheath, which is arranged in a helical pattern along the longitudinal fibers of the tail, wraps around the mid-piece, supplying energy for the metabolic activities o f the sperm. The structure o f the sperm tail is very similar to that of flagella or ci l ia. It is composed o f nine pairs microtubules that are arranged radially around two central filaments (Saacke and Almquist, 1964b; Sull ivan, 1978). A schematic illustration of bul l spermatozoa is shown in figure 2.1. 2.1.1.1. The sperm plasma membrane (SPM) The sperm plasma membrane is a mosaic structure o f fluid lipids and globular proteins, which have a very distinctive l ipid composition (Martinez and Morros, 1996). Its uniqueness is mostly due to highly unsaturated plasmalogens (a kind of ether-linked lipids) that may contribute to the formation of non-diffusable membrane regions or domains capable o f regionalizing both lipids and proteins (Martinez and Morros, 1996). Sperm plasma membrane plays a central role in cellular physiology, cell-to-cell communication, recognition and immunobiology (Douglas and Zuckerman, 1976). Freeze fracture studies on mammalian S P M have shown the presence o f intramembranous particles (IMPs), which are capable of aggregation at specialized sites such as the gap junction, and of reversible aggregation under a variety o f conditions (Friend and Fawcett, 1974; Suzuki, 1990). Human spermatozoa have unusually high levels of unsaturated fatty acyl groups. A lso it contains a variety of phospholipids and fatty acids (saturated- unsaturated) and sterols. The ratio of cholesterohphospholipid is around one in human S P M and this high cholesterol content seems to play an important role in capacitation. Cholesterol is more abundant (4-fold) over 6 acrosomal cap region than over the post-acrosomal segment. Moreover, the inner fusogenic leaflet o f the plasma membrane over the acrosome is relatively rich in free sterols (Friend, 1982). 2.1.1.2. The Sperm Head The nucleus and acrosome are the major components o f the sperm head. It also contains cytoskeletal structures and small amounts of cytoplasm (Eddy, 1988). The oval nucleus containing the highly compact chromatin is enclosed by a non-porous nuclear envelope. The two membranes of the nuclear envelope lie only 7-10 nm apart (Eddy, 1988). The inner surface o f the nuclear envelope referred to as the nuclear lamina, which is comprised almost entirely of deoxyribonucleic acid ( D N A ) condensed by specific nuclear proteins known as histones. This inner surface is thought to form the skeletal framework o f the nuclear envelope and to serve as an anchoring site for chromatin (Garner and Hafez, 1993). The chromosome number and the D N A content o f the nucleus in sperm is half that o f somatic cells o f the same species. The reduction division produce two distinct types o f spermatozoa as far as sex chromatin is concerned. Sperm containing the X chromosome produce female embryos, whereas those containing the Y chromosome produce male embryos (Garner and Hafez, 1993). 2.1.1.3. The acrosome The anterior end of the sperm nucleus is covered by the acrosome, a thin, double-layered membranous sac that is adjacent to the nucleus during spermatogenesis (Garner and Hafez, 1993). The acrosome originates from the Golg i complex in the spermatid, and has two segments, acrosomal cap (anterior acrosome) and equatorial segment (posterior acrosome) (Fawcett, 1975). 7 The acrosome contains a number o f hydro lytic enzymes such as acrosin, hyaluronidase, corona-penetrating enzyme and several acid hydrolases (Morton, 1975) and is involved in the fertilization process. A comprehensive list o f acrosomal enzymes has been presented by Eddy and O'Brien (1994). During fertilization the spermatozoa undergo A R in which the enzymatic contents o f the acrosome are released. The released hyaluronidase disperses the cumulus cells that surround the newly ovulated ova, whereas corona-penetrating enzyme provide a means for the penetrating spermatozoon to pass through the corona radiata o f the ovum. Acrosin, a trypsin-like proteinase, digest a pathway through the zona pellucida for the penetrating spermatozoon (Garner and Hafez, 1993). Acrosin and hyaluronidase, which are major constituents o f the acrosome, are enzymes present in the mammalian sperm acrosome. Acrosin is present in the acrosome as pro-acrosin, which is converted to the active form during A R . Glycosaminoglycans are known to help in the conversion of pro-acrosin (Garner and Easton, 1977). Since acrosin is rapidly released following A R , it has been suggested that the bulk o f the acrosin may be in the soluble acrosomal matrix (Yanagimachi, 1988). 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 (i.e. proacrosin) to active forms (e.g. acrosin), before or during the A R (Yanagimach, 1988). The inner acrosomal membrane ( IAM) is closely applied to the anterior part of the nuclear envelope. The outer acrosomal membrane ( O A M ) surrounds the remainder o f the acrosome and underlies the S P M . Olson et al. (1985) found that lectins bind the inner surface of the O A M and suggested that glycosylated molecules at the site may help to stabilize the membrane or play a functional role in membrane fusion events of A R . The development of the I A M begins during the early spermatid stage of differentiation. It tightly associates with the condensing sperm nucleus, and becomes exposed following the A R (Huang and Yanagimachi, 8 1985). The I A M 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). Localization of glycoprotein molecules on the I A M of guinea pig sperm has been shown by lectin binding studies (Schwarz and Koehler, 1979). Freeze-fracture studies indicate that I A M is very rich in dense intramembranous particles (IMPs), and it has been proposed that the crystalline array o f these particles may represent zona lysin material (Koehler, 1975). The I A M does not directly fuse with the oolema but rather is engulfed into the egg in a phagocytic manner and eventually disintegrated (Bedford, 1972). 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 I A M from fusing directly with the egg plasma membrane (Huang and Yanagimachi, 1985). Two theories have been presented to explain mammalian sperm penetration, enzymatic digestion and mechanical forces (Yanagimachi, 1981). Green and Purves (1984), suggested that the mechanical force o f the motile sperm in conjunction with the visco-elastic properties o f the zona pellucida are sufficient to achieve zona penetration even without the aid of enzymes. 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 possibility and suggests that the rigid nature o f the I A M may be crucial for successful sperm penetration. 2.1.1.4. The flagellum or tail The tail is composed of the neck and the middle, principal, and tail pieces (Garner and Hafez, 1993). The neck or connecting piece is slender and easily fractured. The main structural features of the mammalian sperm tail, are the axoneme, the mitochondrial sheath, the outer dense fibers and the fibrous sheath. The axoneme of the mammalian sperm tail extends 9 the full length of the flagellum. The axoneme consists of two central microtubules surrounded by 9 microtubule doublets arranged radially. These microtubules are composed o f a - and p-tubulin, closely related proteins o f about 55 K D a each. Eddy and O'Brien (1994b) and Saacke and Almquist (1964b) presented detailed reviews on the ultrastructure o f the sperm tail. In the middle piece the 9 + 2 arrangement of filaments is surrounded by nine coarse or dense fibers that appear to be associated with the nine doublets o f the axoneme. These outer coarse fibers possess both elastic and contractile proteins (Garner and Hafez, 1993). The entire middle piece is covered by a mitochondrial sheath, which is arranged in a helical pattern around the longitudinal fibers of the tail, and is responsible for the energy supply o f the sperm. The principal piece, extends to near the end o f the tail. The principal piece is composed centrally o f the axoneme and its associated coarse fibers, a tough fibrous sheath surrounds this central core. The fibrous sheath provides stability for the contractile elements o f the tail (Garner and Hafez, 1993). The end piece is the portion of the tail posterior to the end of the fibrous sheath. It contains only the central axoneme covered by the S P M . 2.1.2. Acquisition of sperm fertilizing ability Spermatozoa are not fertile when they leave the testis. Despite the absence o f gross morphological changes, sperm acquire the ability to interact with and to fertilize eggs during epididymal transit (Eddy et al, 1985; Yanagimachi, 1988; Lacham and Trounson, 1991; Fournier-Delpech and Thibault, 1993). Only when they undergo extensive changes within the plasma membrane and in their motility and metabolism in the female genital tract, this potential fertility comes to action (Fournier-Delpech and Thibault, 1993). 10 The epididymis is adjacent to the testis. Its length is 7 meters in man and 40 meters in bulls. Its main functions are, transport, storage, and functional maturation o f spermatozoa. Time of spermatozoa passage through the epididymis varies with species. In the bul l , it takes 14 days, and in the human it takes 12 days (Fournier-Delpech and Thibault, 1993). During this time period of epididymal transit, spermatozoa undergo some structural and functional changes. Physiochemical and morphological changes in epididymal spermatozoa are collectively referred to as maturation (Austin, 1958; Yanagimachi, 1988; Saling and Storey, 1979). During the maturation process, spermatozoa nucleus develops a resistance to decondensation (Fawcett and Phil l ips, 1969; Fournier-Delpech and Thibault, 1993; Garner and Hafez, 1993). The number o f disulfide-bonds increases in sperm nucleoproteins increases (Esnault and Nicol le, 1976). On the whole, increasing sperm head rigidity protects sperm nucleus and facilitates oocyte zona pellucida penetration. Acrosome becomes more tightly applied to the nucleus (Fournier-Delpech and Thibault, 1993). The plasma membranes seems to undergo molecular changes by inducing new antigenic properties (Jones, 1989) and domains, helping sperm to gain its fertilizing ability (Fournier-Delpech and Courot, 1987). The total l ipid content of the sperm cell also decreases during maturation but the molar ratio o f sterol to phospholipid increases (Believe and Obrien, 1983; Parks and Hammerstedt, 1985). Unsaturated fatty acid content like phosphatidyl choline shows a relative increase during sperm maturation process (Fournier-Delpech and Thibault, 1993). Some changes in protein content of spermatozoa take place including, both addition and removal o f surface proteins (Bedford, 1975; Bedford and Cooper, 1978; Young et al, 1985; Eddy, 1988; Yanagimachi, 1988). Furthermore, new proteins o f low molecular weight originating from accessory gland products may appear on the surface of ejaculated spermatozoa (Young et al, 1985), acting to change charge density and adhessiveness (Believe and O'brien, 1983). In support o f these findings, a gradual major change in the distribution o f lectin binding 11 sites between caput and cauda epididymal spermatozoa have been reported (Friess and Sinowatz, 1984). A l l these changes contribute to rendering the spermatozoa potentially fertile and A R resistant, as necessary, for transport and storage. It is only after reaching the epididymal body that spermatozoa become able to move linearly. Before that, in the caput epididymis, spermatozoa are motionless (Fournier-Delpech and Thibault, 1993). Even by increasing cyclic adenosine mono phosphate ( cAMP) content or by caffeine treatment o f caput spermatozoa, no linear progression could be induced. Proteins secreted by epididymis (forward motility proteins or F M P ) are essential to induce progressive motility (Serres and Kann, 1984) especially in bovine spermatozoa (Acott et al., 1983). In general, spermatozoa removed from cauda epididymis w i l l fertilize egg in-vitro much more readily than ejaculated spermatozoa. Epididymal spermatozoa become infertile once they are exposed to seminal plasma. Some proteins from accessory glands secreted into the seminal plasma are responsible for their inability to fertilize eggs. They bind to spermatozoa and prevent them from undergoing capacitation and acrosome reaction (Mi l ler and A x , 1990; Nass et al., 1990; Manjunath et al., 1993a). These surface factors are described as "decapacitation factors" (Chang, 1957). Decapacitation factors should be either removed or masked for spermatozoa to be able to gain fertilizing ability (Hunter and Nornes, 1969; Eddy, 1988; Yanagimachi, 1988). During sperm transport in the female genital tract or in a defined medium under in vitro condition, decapacitation factors can be removed (Hunter and Nornes, 1969; Eddy, 1988; Yanagimachi, 1988). 12 9 10 '3 H 11 PLATE 2.1. Schematic structure o f the mammalian spermatozoon including: 1) Sperm plasma membrane 2) Outer acrosomal membrane 3) Acrosomal content 4) Inner acrosomal membrane 5) Nuclear membrane 6) Nucleus 7) Neck 8) Mitochondrial sheath 9) Annulus 10) Principal piece 11) End piece. 13 2.1.3. Sperm surface domains Mammal ian spermatozoa segregate their organelles to specific intracellular locations where they function at defined fertilization steps. There are four major subdivisions of the spermatozoa, determined by specific organelles. They are, acrosomal (anterior acrosome or acrosomal cap and equatorial segment or posterior acrosome) and post acrosomal segments of the head and the midpiece and principal piece of the tail. The domains are dynamic features that undergo changes in organization and composition during the life of the spermatozoa (Eddy, 1988). Most surface domains are established during spermatogenesis while the round spermatid is being remodeled into the spermatozoon (Eddy, 1988). New surface antigens also appear in specific domains during maturation (Eddy and O'brien, 1994). During fertilization, specific domains o f sperm head membrane function in the binding and penetration o f the zona pellucida and in the fusion with the egg plasma membrane (Yanagimachi, 1988). For example, receptors for the zona pellucida are localized in the periacrosomal domain. Upon their interaction with zona, membrane fusion A R is initiated (Wassarman, 1987). In contrast, the postacrosomal plasma membrane function after sperm penetration into zona and represents the site o f initial fusion with the egg plasma membrane (Yanagimachi, 1988). Thus the molecular and structural heterogeneity of the different sperm membrane domains relates to their restricted function in fertilization. During the three stages of epididymal maturation, capacitation, and acrosome reaction, numerous surface changes are happening including, changes in localization, level of surface expression, molecular structure, and lateral mobility. Carbohydrate moieties are distributed with a regional heterogeneity on the sperm surface, as indicated by previous lectin-binding studies (Edelman and Mil lette, 1975; Ahluwal ia et al., 1990). Large numbers o f lectin-binding sites have been identified on 14 spermatozoa (2.5 x 10 6 to 4.9 x 10 7 sites/cell), occuring with higher density on the head, than on the tail (Edelman and Millette, 1975). 2.2. Sperm capacitation During epididymal transit, sperm gain the ability for progressive motility, zona-binding, and fertilization (Fournier and Thibault, 1993). Cauda epididymal or ejaculated spermatozoa are potentially fertile. However, due to the presence o f decapacitating factors, their fertilizing ability is temporarily lost at ejaculation. Spermatozoa must reside in the female reproductive tract (in vivo) or in a defined medium (in vitro) for a certain period of time, during which the sperm undergo a series of physiological changes. Such physiological changes that render the spermatozoa competent for fertilization are collectively called capacitation (Austin, 1951; Chang, 1951). Capacitation is a reversible phenomenon, as capacitated spermatozoa can be decapacitated by re-introducing them to seminal plasma. Capacitation is not species-specific: bull sperm w i l l capacitate in ewe and sow; rabbit sperm in rat or mouse or hamster, or even in bitch; mouse sperm in rat and hamster (Fournier and Thibault, 1993). The experimental evidence for capacitation in the rabbit and rat was first documented by Chang (1951) and Austin (1951). It has been suggested that the ampulla is the capacitation site (Yanagimachi, 1988). In vivo, the time required for capacitation depends on the oestradiol/progesterone concentration ratio. The duration o f capacitation is the shortest at the end o f the ovulation process (Yanagimachi, 1988). Capacitation does not occur in the uterus during the full luteal phase (cow, pseudopregnant rabbit) (Fournier and Thibault, 1993). Capacitation seems to be necessary in all mammalian species, at least with ejaculated spermatozoa (Fournier and Thibault, 1993). However, in some species like the human, capacitation time is very short (Fournier and Thibault, 1993). When the time required for 15 sperm liquefaction (20-30 min), seminal plasma removal, and sperm attachment as wel l as its complete penetration, are taken into consideration, it seems that human sperm capacitation needs only few minutes (Plachot et al, 1986). In vivo and in vitro capacitation time o f ejaculated spermatozoa have been reported for bulls as 8 and 6 hours, respectively (Foumier and Thibault, 1993). In human, the in vitro capacitation time for spermatozoa is 2 hours (Lee et al., 1998; Garner and Hafez, 1993). Specific lectins or antibodies do not uniformly attach to all spermatozoa. This suggests a heterogeneity in the sperm population. The few l iving spermatozoa present in the ampulla are all capacitated and able to fertilize oocytes immediately, it can be concluded that a selection o f a small number o f "capacitable" spermatozoa may have occurred along the female reproductive tract. This may explain the necessity o f high numbers o f spermatozoa to obtain high in vitro fertilization rates (Fournier and Thibault, 1993). 2.2.1. Biological and molecular changes in spermatozoa during capacitation During ejaculation, compounds originating from secretions o f the accessory glands adhere to the sperm plasma membrane. Removal o f the seminal plasma and its components, referred to as decapacitation factors, is a requirement to facilitate capacitation (Yanagimachi, 1988; Manjunath et al, 1993a). Removal of complexed glycosyl residues result in a decrease in the glycoproteins molecular weights, mefhylation o f phospholipids, decreases of sterol/phospholipid ratios, increase in lateral mobility of lipids and proteins (O'Rand, 1979; Oliphant and Singhas, 1979; Sidhu and Guraya, 1989). The respiratory activity o f sperm increases, perhaps due to the presence of energy substrates in the capacitation medium. However, a rise in intracellular p H reflects ion exchanges, proton efflux, a rise in membrane potential, and a decrease in K+ cellular content (Fournier and Thibault, 1993; Vredenburgh-Wilberg and Parrish, 1995; Zeng et al, 1995). 16 It has been shown that an influx of extracellular C a 2 + is essential for A R to happen (Yanagimachi and Usui , 1974; Singh et al, 1978; Kazazoglou et al, 1985; Fraser, 1987). Calmodulin, an acidic, low molecular weight protein, regulates 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 S P M (Manjunath et al, 1993b) and participates in the regulation of C a 2 + transport during capacitation and A R . Heparin (in vitro) and heparin-l ike substances found in the female reproductive tract (in vivo) may influence the calcium regulatory role o f calmodulin through their interactions with bovine seminal plasma proteins. Act in polymerization has been reported also to be directly involved in capacitation and translocation o f the membrane proteins (Saxena et al, 1986b). 2.2.1.1. Protein changes during capacitation According to the physical relationship between proteins and the plasma membrane, three different categories of changes are possible: 1) Non-covalently bound proteins are leached, when spermatozoa are incubated either in uterine/tubal fluids from estrous females, in vivo or in a culture medium, in vitro (Fournier and Thibault, 1993). 2) A decrease in molecular weight o f structural proteins or epididymal proteins caused by the losses of sugar moieties during sperm capacitation. This phenomenon was demonstrated by temporal and local changes in lectin binding (Fournier and Thibault, 1993). 3) Migration o f some proteins in S P M can take place in short distances or in very small areas (Vil larroya and Scholler, 1987). Protein migration in very small areas make particle-free and particle-rich domains in the sperm head plasma membrane, which is a well-known preliminary step to apposed membrane fusion, and outer acrosomal membrane. Fusion between the plasma 17 membrane and outer acrosomal membrane w i l l begin in particle-free areas (Suzuki and Yanagimachi, 1989). 2.2.1.2. Lipid content and ratio changes during capacitation The plasma membrane of ejaculated sperm contains a high proportion o f cholesterol and sterol sulfates. In bulls, the molar ratios of cholesterol/phospholipids for plasma membrane, outer acrosomal membrane and sperm total lipids are 0.38, 0.26 and 0.26, respectively. The first two ratios decrease during capacitation due to the loss o f free and conjugated sterols. Since cholesterol molecules are very rigid and prevent flexing of l ip id carbohydrate chains, their release from plasma membrane results in an increases o f membrane fluidity during capacitation (Davis, 1981; Langlais and Roberts, 1985; Ehrenwald et al, 1988a, 1988b). When liposomes containing cholesterol or cholesterol sulfate are introduced into incubation medium containing capacitated spermatozoa, they are decapacitated (Ehrenwald, 1988a, 1988b). If the decapacitated spermatozoa are resuspended in cholesterol-free medium, they partially lose the previously incorporated cholesterol (30%), and i f replaced in a capacitating medium, they are able to fertilize (Ehrenwald, 1988a, 1988b). Studies have confirmed the importance of cholesterol acceptor molecules (such as B S A or blood serum) in the capacitation medium for successful capacitation to take place (Sidhu and Guraya, 1989). A lbumin, a multiple ligand carrier, mediates sperm cholesterol efflux during in-vitro capacitation in many species including human and bul l (Ehrenwald et al, 1988a; Langlais et al, 1988; Parrish and First, 1991). Several other factors have been identified as cholesterol acceptor molecules including very low ( V L D L ) , low (LDL) , and high density lipoproteins (HDL) from serum and follicular f luid (Langlais et al, 1988). In the absence of cholesterol acceptors, or in the presence of an agent such as glucose that would prevent cholesterol acceptance, capacitation cannot occur (First and Parrish, 1987a, 1987b; Florman and First, 1988; Parrish et al, 1989). 18 2.2.1.3. Changes in sperm motility characteristics Mature spermatozoa recovered from the cauda epididymis or ejaculated spermatozoa, possess a forward (progressive/linear) motility. A s shown in micro-cinematographic studies, a weak asymmetry in flagellar beating induces a slightly curved progression which changes regularly from right to left and vice versa. The combination o f flagella movement and the 180° head rotation gives the impression of a straight progression. Fol lowing capacitation, two changes occur. A greater flexibility of the flagellum, mainly of the midpiece, followed by an increased beating amplitude, cause a whiplash type of sperm movement. Consequently, the sperm head swings slightly in opposition to the flagellum and head rotation ceases so that progression becomes circular (Yanagimachi, 1981; Fournier and Thibault, 1993). These changes in the motility patterns of capacitated sperm are collectively called hyperactivity. Capacitation-related changes in motility patterns also occur in bull spermatozoa. However, they display a different form of hyperactivity as compared to what is typically seen in other laboratory animals (Mcnutt, 1990). During capacitation, there is a gradual rise in intracellular C a 2 + (Fournier and Thibault, 1993). Hyperactivation is more directly related to c A M P levels than to C a 2 + influx. A progressive increase in Ca 2 +-dependent c A M P level precedes the onset o f hyperactivated motility (Fournier and Thibault, 1993). 2.2.2. Relationship between capacitation and the fertilizing ability Capacitation is an essential pre-requisite for the occurrence o f the A R and fertilization of the oocytes. Under in vitro conditions, spermatozoa need to undergo a succesful capacitation and A R in order to accomplish fertilization. On the other hand, unsuccessful in vitro fertilization, can happen by reasons other than failure o f spermatozoa to be capacitated 19 (Yanagimachi, 1988). Thus A R also can be taken as a reliable indicator o f capacitation (Parrish et al, 1988; Yanagimachi, 1988). During capacitation, a gradual loss o f coating proteins or rearrangement and processing of structural proteins are known to take place in sperm plasma membrane. This allows sperm receptors to be fully functional. On the other hand the increase in membrane fluidity would facilitate the binding o f zona pellucida proteins to sperm receptors (Fournier and Thibault, 1993). Final ly these changes would result in high levels of recognition specificity between sperm and zona. In addition, formation of particle-free areas and changes in l ip id composition and ratio during capacitation stage, are essential for acrosome reaction (Fournier and Thibault, 1993). Hyperactivation is one of the major changes in capacitation, which is not necessary to traverse the cumulus mass. It has been shown that incapacitated spermatozoa as wel l as capacitated ones penetrate to the cumulus cells. However, the strong beating force generated by hyperactivation is essential for the spermatozoa to pass through the zona. Scanning electron . microscopy has shown that lateral head swaying slightly slits the external layer o f the zona (Fournier and Thibault, 1993). Fol lowing capacitation, several changes in the post-acrosomal plasma membrane occur. These changes are essential prerequisite for sperm/oocyte fusion. But still capacitation by itself is insufficient to allow gamete fusion (Fournier and Thibault, 1993). 2.2.3. Capacitating ability Around the ovulation period, special physiological characteristics o f the uterine mil ieu such as a low p H = 6-0, ewe, cow and rabbit doe), a low protein content, and proteolytic and glycosidic activities seem to favor protein removal and protein processing. A strong sterol sulfatase activity (women, hamster) liberates free cholesterol that can be easily removed from 20 the sperm membrane by cholesterol acceptors such as albumin and lipoproteins ( H D L , L D L , V L D L ) , which are present in uterine and oviductal fluids (Fournier and Thibault, 1993). In the cow, H D L content is high when serum progesterone is low (follicular phase and ovulation) and vice versa in the luteal phase. Moreover, in the follicular phase, sterols/phospholipid ratio is low (0-5 vs > 2 in blood). In the luteal phase, by contrast, the low potential o f the uterus to capacitate sperm may be explained by both a high cholesterol phospholipid ratio (= 2) and a lower lipoprotein content. 2.2.4. Capacitation In Vitro Sperm capacitation has been studied in all mammalian species. Effective capacitation conditions in vitro mimic those existing in vivo: In vitro removal of seminal fluid and incubation o f washed spermatozoa in capacitation medium are essential for sperm survival and capacitation. Some media mimic tubal medium or blood plasma (TC 199). Generally, they originate from traditional culture media such as Tyrode's and Kreb's-Ringer's with small variations. The presence o f cholesterol acceptor molecules, blood serum or serum albumin (bovine B S A or human H S A ) can induce capacitation. Blood serum is more efficient than albumin because it contains lipoproteins (HDL , L D L , V L D L ) that are better acceptors than albumin (11%, 40% and 44%, respectively, against 7%). Foll icular f luid containing H D L and albumin is also an effective sterol acceptor (Fournier and Thibault, 1993). The presence o f heparin in the capacitating medium has been reported to effectively increase in vitro fertilization rates in cattle (Handrow et al, 1984). Since it has been shown that glycosaminoglycans, including heparin, are secreted in the cow and ewe uterus and 21 fallopian tubes, the capacitation inducing effect seems to be physiological (Handrow et al, 1984). Furthermore, certain in vivo conditions which seem essential for membrane protein release or processing may not necessarily be delivered in vitro, such as low p H , and proteolytic or glycosidasic activities. However, as capacitation occurs, a high sperm concentration is always necessary to obtain capacitation in vitro. Experiments with boar spermatozoa are particularly suggestive of the importance of a high concentration o f spermatozoa to obtain capacitation in vitro (Nagai et al, 1984). Since during incubation as many as, 10% or more spermatozoa may become spontaneously acrosome-reacted, acrosomal enzymes released into the medium in sufficient amounts (that depends on sperm concentration), may modify membrane protein composition and speed up the capacitation process. This hypothesis is supported by the fact that addition of trypsin to incubation medium can speed up sperm capacitation in rabbit, hamster, and bull . However, human sperm capacitate under relatively low concentrations (1 x 10 5 sperm/ml). Protease and plasmin activities, which are responsible for sperm liquefaction, may be relevant factors to reduce capacitation time of human spermatozoa. On the other hand, in vitro, proteins secreted in the uterus and tubes that coat the sperm plasma membrane and protect it are missing. This is one of the explanations for the difficulty in prolonging in vitro survival over 24-36 hours, even in species in which spermatozoa survive in vivo for days or weeks. Although some factors such as adrenaline and taurine/hypotaurine present in vivo are known to prolong sperm motility when added to the capacitation medium (Yanagimachi, 1982), the environmental conditions which allow a long survival in vivo at 37-39°C have not yet been clarified. 22 2.3. Sperm acrosome reaction The acrosome is a baglike structure consisting of an inner and an outer acrosomal membrane present at the anterior side of the head covered by plasma membrane. The mammalian A R is essential for fertilization and occurs in sperm that have already undergone capacitation. Three distinct morphological stages of A R , acrosome swelling, vesiculation, and shedding, have been identified (Sidhu et al., 1986) resulting in the display o f a new cel l surface domain in the apical region of the sperm head (Florman and First, 1988). The A R allows release o f the hydrolytic enzymes required for penetration o f sperm through egg investments and related steps, and appears to modify the equatorial segment for attachment with the egg membrane (Bedford, 1983). Maintaining a high intracellular concentration (140 m M ) of K + , low concentration (5-10 m M ) of N a + , and a very low concentration (0.15 u,M) o f C a 2 + is important for preventing the spermatozoa from undergoing premature A R . This is accomplished, at least partly, by the N a + / K + ATPase (pumping N a + and K + into the cell) and C a 2 + ATPase (pumping C a 2 + out of the cell) systems. The A R and exocytosis of somatic exocrine cells are similar in regard to the requirement of C a 2 + influx and clearing of intramembranous particles at the exocytotic sites. However, A R differs from somatic cell exocytosis in that (Robaire and Hermo, 1988) it includes loss o f the membranes unlike in somatic cell exocytosis and (Setchell and Brooks, 1988) in A R the exocytotic sites become cholesterol-poor whereas Orci et al. showed in pancreatic acinar cells that exocytotic sites contain cholesterol. Mammal ian A R is distinguished by two additional characteristics: (Robaire and Hermo, 1988) it does not appear to require some specific stimulus, but an adequate level of Ca in a physiological mil ieu once capacitation is completed, and (Setchell and Brooks, 1988) there is little synchrony of the reaction among the spermatozoa in a medium. The observation that capacitation is not 23 necessary i f C a z + is driven by other means (Yanagimachi, 1975, Fraser et al, 1995) indicates that the primary role o f capacitation is to regulate the timing of C a 2 + influx. Since a successful fertilization requires the functional significance of A R , it would not be realistic to consider any site in the female reproductive tract other than immediate vicinity of the oocyte for A R induction. Thus ampulla, as the site o f fertilization would be the best place for A R . In contrast, uterus is too early a site for the occurrence of A R (Sidhu, 1989). But studies have shown that even locations such as the uterus (Wooding, 1975; Sidhu et ah, 1986), far away from the exact site of fertilization, definitely play a role in the in the induction o f A R in bovine spermatozoa. Acrosome-reacted spermatozoa can not survive long enough to reach the oocyte in ampulla while they are alive. Thus A R occurring in distant locations such as the uterus may not really be of any functional significance to the fertilization, instead, it could be one of the mechanisms of removing surplus spermatozoa from the competition for fertilization and consequently deceasing the chance for polyspermy. It is wel l established that A R could be induced by either specific factors or by cell-cell interactions (Mcnutt, 1990; Ell ington et al, 1991; Guyader and Chupin, 1991; K ing et al., 1994). Previous studies indicate several factors in female reproductive tract to be involved in the induction o f A R . Among these factors zona pellucida (Meizel , 1985; Cross et al, 1988; Florman and First, 1988; Meize l et al, 1990; Carrell et al, 1993), follicular f luid (Meizel , 1985; Tesarik, 1985; Suarez et al, 1986; Yud in et al, 1988), cumulus cells (Meizel , 1984, 1985; Tesarik, 1985; Cross et al, 1988a,c), glycosaminoglycans present in cumulus cells (such as heparin) (Lenz et al, 1982,1983; Handrow et al, 1984; Parrish et al, 1985; A x et al, 1985) have been mentioned. Based on information presently available, C a influx occurs during capacitation and as a consequence of glycosaminoglycans (present in cumulus cells) or glycoproteins o f zona pellucida. It has not been clearly established how this influx is initiated. It has been suggested 2 4 that the glycosaminoglycan-receptors on sperm plasma membrane may be C a carriers which upon activation can facilitate the diffusion of extracellular C a 2 + (Yanagimachi, 1990). On the other hand G A B A A (Gamma-aminobutyric acid type A ) receptor/Cl" channels are believed to be activated by progesterone, which is present in follicular f luid and cumulus cells (Bormann and Clapham, 1985; Mul ler et al, 1989; W u et al, 1990; Burt and kamatchi, 1991; Majewska, 1992; Zhang and Jackson, 1993; Grover et al, 1993; Ka i la , 1994; Hales et al, 1994; Bonnet and Bingmann, 1995). Eff lux of CI" and as a consequence a decrease in electronegativity of sperm membranes ensure the opening of gradient-sensitive C a 2 + channels. A n influx o f C a 2 + is then the end result which turns off N a + / K + ATPase pump, leading to an influx of Na + " Increasing N a + can then be used by C a 2 + / N a + antiporter, which in turn increase the C a 2 + concentration inside the sperm. A n efflux of H * and intracellular p H increase is the next step. On the other hand C a 2 + entering the sperm, acts on the plasma membranes and outer acrosomal membrane and activates the membrane-bound phospholipase A2 and C. These enzymes are active at alkaline p H ensured by H + efflux. These phospholipases act then on phospholipids in the membrane, leading to the release of fusogenic lysophospholipids, fatty acids and diacylglycerols. Sperm plasma membrane and outer acrosomal membrane fusion in effect o f the fusogenic lysophospholipids happens ensuring the membrane vesiculation and C a 2 + entry to the acrosomal matrix. Fol lowing C a 2 + influx and H + efflux in acrosomal cap, proacrosin is converted into acrosin, the active version. Acrosin is a trypsin-like glycoproteinase which is found within the acrosome. The majority of acrosin exists in an inactive form, called proacrosin (Meizel , 1984). The acrosomal membrane is dispersed by this active enzyme and the released acrosomal content (various enzymes) lead to acrosome reaction. Sperm either should attach to the oocyte or die very soon, since the energy consumption increases during capacitation and A R and A T P deplete soon after A R (Fournier-Delpech and Thilbault, 1993). Moreover the excessive C a 2 + within the cell are detrimental to sperm (Yanagimachi, 1990). 25 2.4. Structure of mammalian oocyte The oocyte is a highly differentiated cell , which is capable o f being fertilized. A schematic illustration of oocyte and its associated cells is presented in Plate 2.2. Before ovulation oocyte is at one side o f foll icle, embedded in a solid mass o f follicular cells, cumulus oophorus. Recently ovulated oocytes surrounded by a variable number o f granolosa cell layers (corona radiata) and a matrix o f follicular fluid. After ovulation cumulus and corona radiata cells are present only for a few hours (Thomson and Wales, 1987). The in vitro exposure of recently ovulated oocyte to oviductal fluids containing fibrinolytic enzymes results in retraction and degeneration o f these cells. Two different membranes cover the oocyte, vitelline membrane with the properties o f plasma membrane in somatic cells (diffusion and active transport) and zona pellucida, a homogenous and semi-permeable membrane. Zona pellucida is composed o f a conjugated protein so it can be dissolved by proteolytic enzymes such as trypsin and chymotrypsin. Several benefits have been reported for oocyte membranes including, protection of the oocyte and selective absorption of inorganic ions and metabolic substances. Vitel lus comprises most of the volume within the zona at the time of ovulation. After fertilization, it shrinks forming a perivitelline space between the zona and the vitelline membrane. Polar bodies are also situated in this space. Vitel lus structure is species-dependent mainly because o f varying amounts of yolk and fat droplets. In goat and rabbit oocytes, yolk granules are finely divided and uniformly distributed so that nuclear changes are visible. In horse and cow, oocyte is fil led with fatty and refractile droplets (dark mass o f vitellus) so that inside o f the oocyte is not visible. If oocyte fail to become fertilized, the vitellus fragments into units o f equal size, each containing one or more abortive nuclei. Ful ly-grown oocytes contain round or oval, highly vacuolated mitochondria that have arranged crista (Zamboni, 1972). A s in the case o f mitochondria, the Golg i complex undergoes 2 6 marked ultrastructural changes during oocyte growth (Zamboni, 1972). In the late stages of oocyte growth, Golg i complexes exhibit increased numbers of very swollen, stacked lamellae that are associated with numerous vacuoles, granules, and l ipid vesicles. These changes are consistent with increased participation of the Golg i complexes in the processing and concentration o f secretory products and cortical granule formation during oocyte growth (Zamboni, 1972). Cortical granules formation can be regarded as a feature o f oocyte growth and differentiation (Kruip et al, 1983; Cran, 1987, 1989). These granules fuse with the vitelline membrane at fertilization and by releasing their contents into the perivitelline space, alter the functional properties o f the zona pellucida. Cortical granules are small, spherical, membrane-bound organelles, which are thought to resemble lysosomes. Oocyte growth is accomplished by the formation and accumulation o f increasing numbers o f the granules (Kruip et al, 1983; Cran, 1987, 1989; Cran and Esper, 1990). During the growth phase of oocyte, the number of ribosomes present in the cytoplasm increases. The changes in ribosome population during oocyte growth is believed to be related to changes in rates of protein synthesis during this period. Many biochemical changes are known to occur during the growth of the oocyte. Carrol l and Whittingham (1993) have reported that the mechanisms of C a 2 + homeostasis in oocytes change during growth and maturation. During their growth, primary oocytes are known to synthesize a variety of proteins. The general pattern of protein synthesis is believed to remain constant throughout oocyte growth, but there are changes in the relative rate of synthesis o f specific proteins (Carroll and Whittingham, 1993). The plasma progesterone concentration remains very low during follicular development and only after L H surge, the progesterone level starts to increases (Kruip and Dieleman, 1985; Dieleman and Bevers, 1987; Dieleman and Blankenstein, 1987). The maturation of the oocyte 27 is l ikely to be influenced by the change in the estradiol/progesterone balance within the follicle (Kruip and Dieleman, 1985; Dieleman and Bevers, 1987; Dieleman and Blankenstein, 1987). A number of workers have reported on the morphology o f bovine oocytes after in vitro maturation (Sirard and Lambert, 1985, 1986; Lambert et al., 1986). The ideal morphology is described as an oocyte with an expanded and clear cumulus cell mass. Schellander et al. (1989) assessed quality in oocytes recovered after in vivo maturation as excellent when the ooplasm was evenly granulated, the perivitelline space small and the cumulus cells fully expanded without evidence of degenerated cells. 2.5. Fert i l izat ion 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). In most mammals, fertilization begins after the first polar body has been extruded and the second reduction division is in progress (metaphase II o f the second meiosis). In contrast, in the horse and dog, the sperm enter before the second reduction division begins (Baser et al., 1993). Three different steps are required for mammalian oocyte to be fertilized, Sperm migration through cumulus cells, sperm attachment and migration through zona pellucida, and sperm/oocyte plasma membrane fusion (Baser et al., 1993). 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 o f the spermatozoon, leading to its tight binding (Wassarman, 1990). Penetration o f the zona by spermatozoa occurs within 5-15 minutes after the binding. A n acrosome-intact sperm is essential for attachment (Wassarman, 1990). Fol lowing sperm binding with Z P 3 , interactions of sperm with other components o f zona (ZP2) and consequently A R happen (Wassarman, 1990). Both sperm motility and acrosomal enzymes are needed for zona penetration 2 8 (Yanagimachi, 1981). Attachment of spermatozoa occurs initially at the equatorial segment of the sperm head. Once into the perivitelline space, the spermatozoon becomes associated with the plasma membrane of the egg and eventually fuses with it. The "cortical reaction" (release of cortical granule material) takes place soon after, preventing the entry o f any further spermatozoa. In deed, cortical reaction results in the release of enzymes that cause hardening of the zona and inactivation o f sperm receptors (ZP3). On vitelline penetration, the activated oocyte completes its meiosis and expels the second polar body into the perivitelline space (Wassarman, 1990). Decondensation o f 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 o f 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 fol low the fusion of the spermatozoon and egg. D N A 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 center o f 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 o f chromosome material can be considered as the end of fertilization and the beginning of embryonic development. Parrish and First (1991) suggest that the bovine fertilization may be similar to that in the mouse. 29 PLATE 2.2. Schematic structure of the mammalian oocyte after ovulation including: 1) Cumulus Cells 2) Corona Radiata 3) Zona Pellucida 4) Perivitelline Space 5) Vitelline Membrane 6) Cortical Granules 7) Cytoplasm 8) Nucleus Membrane 9) Chromatin. 2.6. Laboratory procedures for fertility evaluation Numerous attempts have been made to identify some semen characteristics which can accurately predict the fertilizing potential o f spermatozoa. The major purpose o f semen evaluation is either predicting or enhancing the ability o f the sperm to fertilize an oocyte. Even though the most val id test of sperm fertilizing ability is a viable pregnancy and a normal offspring fol lowing in-vivo insemination (Bavister, 1990), the availability of simple in-vitro tests that can predict sperm fertilizing ability, w i l l be of great practical significance. Some of the important methods currently available are discussed here. 30 2.6.1. The relationship of sperm motility, viability, and morphology with fertility Sperm concentration, motility, viability and morphology estimates are considered the most important among the parameters necessary for a basic analysis o f semen. Nigrosin and trypan blue have been conventionally used for assessing membrane integrity o f bull spermatozoa. The usefulness o f nucleic acid specific stains (bisbenzimide) have been reported for the sorting o f live cells according to their D N A content (Arndt-Jovin and Jovin, 1977). Commercial ly available stains such as Hcechst 33258 and Hoechst 33345 (Sigma, St. Louis, M O , U S A ) have been found useful for assessing membrane integrity o f spermatozoa in a wide variety o f species including human (Cross et al., 1986). Saacke (1970) has reported a significant correlation between post-thaw motility and maintenance o f acrosome in bul l spermatozoa. Direct counts of spermatozoa for intact acrosomes were found to be highly repeatable, with a coefficient of variance (CV) o f 6% in comparison to a C V of 25% for motility estimates. Wil l iams and Savage (1925) found that i f abnormal spermatozoa exceed 18%, the fertility declined. Even though a wide variety of morphological abnormalities of spermatozoa have been reported, there is no clear experimental evidence o f a relationship between specific morphological characteristics and fertility; however, a high frequency of abnormal spermatozoa has been associated with reduced fertility (Sull ivan, 1978). The maximum permissible limit for head abnormalities is set at 5%, and for total abnormalities (all categories o f abnormalities) it is 20%. A n y sample exceeding these limits is considered unsuitable for A I . 2.6.2. The relationship of sperm freezability with fertility Freezing and thawing procedures severely impair the cellular functions o f spermatozoa (Parks and Graham, 1992), resulting in reduced fertility (Hammerstedt et al., 1990). Therefore, post-thaw sperm quality is an important factor in the artificial insemination industry, 31 as it directly related to fertility and exhibits much variability between bulls (Elliot, 1978). Co ld shock index has been tried to assess the resistance o f spermatozoa to low temperatures (Sull ivan, 1978). In this test, 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 motil ity and viability. This test is still valuable in those parts o f the world where chilled semen is used for artificial insemination. The possibility Of identifying specific enzymes that may be damaged during cryopreservation, have been studied in the University of Guelph, Ontario, Canada. The loss o f such enzymes during freeze-thaw process and its effect on fertility have been studied (Buhr and Zhao, 1995) and M g 2 + - A T P a s e , N a + - K + ATPase and C a 2 + ATPase have been found to be affected during cryopreservation o f bull spermatozoa. It has been suggested that such loss or damage o f enzymes during cryopreservation may contribute to the reduced fertilizing ability o f frozen-thawed sperm. 2.6.3. The relationship of sperm biochemical parameters with fertility Metabolic activity measurements in spermatozoa have been considered as possible predictors o f fertility. The use o f metabolic tests such as oxygen uptake (bishop and Salisbury, 1955) and pyruvate oxidation (Melrose and Terner, 1953), have been suggested, but were not found useful for routine evaluation of semen. A kit for rapidly and conveniently assessing sperm viabil ity by measuring A T P loss is now commercially available (Sperm Viabi l i ty Test, FireZyme Diagnostic Technologies Limited, Halifax, N S , Canada). This test uses an enzyme, Luciferase (derived from fire-fly), which oxidizes luciferin (substrate) in proportion to the concentration of A T P present, resulting in the emission of light. Since A T P disappears within seconds fol lowing cell death, only the viable spermatozoa w i l l contain A T P to contribute to the light-producing reaction. The light produced is measured in a bioluminometer. Published 32 information on the usefulness of this test for making fertility estimates is still not available, but it may provide some reliable means of predicting fertility o f semen samples. 2.6.4. The relationship of sperm-zona binding/oocyte penetration assays with fertility Sperm-zona binding or oocyte penetration assays (Bosquet et al., 1983; Graham and Foote, 1987a; 1987b; Wheeler and Seidel, 1987; Boatman et al., 1988; Fazeli et al., 1993, 1995) have been reported as predictors o f fertility. The zona-free hamster egg penetration assay originally described by Yanagimachi (1972) and reported to have some applications as a fertility test in humans. However, since this assay does not measure the ability of sperm to penetrate the zona, it does not always correlate wel l with fertilization in-vitro (Bosquet et al., 1983; Critser and Noi les, 1993). The hemizona binding assay has been reported mainly as a diagnostic test for fertility prediction in humans (Burkman et al., 1988; Fazeli et al. ,1995). Since zona pellucida of individual oocytes have a highly variation in their sperm binding capacity, two matching halves o f zona from the same oocyte allows a minimization of such variation. 2.6.5. The relationship of acrosome reaction with fertility A positive correlation has been reported between the percentages of intact acrosomes and non-returns to first insemination, whereas only a weak correlation was obtained when percentages of motility were compared with non-returns (Saacke and White, 1972). A definite relationship has been reported between fertility and the ability of spermatozoa to acrosome-react under the influence o f heparin, calcium ionophore A23187, and lysophosphatidylcholine (Ax et al., 1985; Parrish et al., 1985a, 1985b, 1985c; A x and Lenz, 1986; Graham and Foote 1987a, 1987b; Whitf ield and Parkinson, 1992). A relationship between the binding affinity of heparin to spermatozoa and fertility has also been demonstrated (Marks and A x , 1985; lalich et 33 al, 1989; Be l l in et al., 1993). A test based on calcium ionophore-induced A R in human spermatozoa has been found useful in identifying semen samples o f sub-fertile/infertile men, indicating acrosomal dysfunction as a l ikely cause o f fertilization failure. This test may have a predictive value for fertility (Cummins et al, 1991). Results of these studies strongly suggest that the ability o f spermatozoa to undergo A R in-vitro may be useful in predicting the fertility o f bulls. 2.6.6. The relationship of in vitro fertility with in vivo fertility Several attempts have been made to correlate the results o f bovine I V F with in-vivo fertility but conflicting results have been obtained (Ohgoda et al., 1988; Hi l lery et al., 1990; Marquant-Le Guienne et al, 1990). It has been reported that 56-60 day non-return rates are significantly correlated with the first cleavage in-vitro, but further embryonic development in-vitro is not correlated with non-return rates (Shamsuddin and Imrsson, 1993). Further investigations are definitely needed to determine i f results o f I V F would qualify as a predictor for the in-vivo fertility o f 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 A R in-vitro. A better understanding of the processes of bovine sperm capacitation and A R in-vitro may be a basis for developing methods to enhance the success rates in bovine I V F and embryo production systems. 2.7. In vitro fertilization Successful cattle in vitro fertilization requires appropriate preparation o f both sperm and oocyte, as wel l as culture conditions which are favorable to the metabolic activity o f the male and female gametes (Brackett, 1981, 1983; Sirard, 1989a, 1989b; X u and K ing , 1990a). Unt i l 3 4 1985 no more than ten I V F calves had been reported in the literature (Brackett et al, 1982, 1984; Sirard et al, 1986a). In all of them, the calf was the result o f fertilization in vitro of an ovulated or in vi'vo-matured oocyte. The first report of successful in vitro maturation and I V F of cattle oocytes was that of Iritani and N i w a (1977) in Japan but the birth o f calves was not reported until the work of Hanada et al. (1986), three calves were born, representing 2 1 % of the embryos transferred to recipient animals. Possibly the largest group o f in vitro maturation and I V F calves born at that time was from the work of L u et al. (1987b, 1988b). In his report, 18 calves were born alive to 13 recipients after non-surgical transfer of embryos; this represented an embryo survival rate o f 59%. The report of L u et al. (1987b) set out information on the methodology employed in the production of embryos but and provided the first convincing evidence of normal pregnancy rates with this type of embryo, as wel l . Normal ly in cattle IVF , frozen semen is used to fertilize the oocytes. Using semen obtained from cattle A I stations implies that sperm must have shown a certain standard o f motility to be accepted for freezing in the first instance. There is stil l, however, the need to double check for satisfactory sperm motility and other characteristics after the sperm thawing. In spite of the long history of the freezing of bull sperm, even with current techniques 50% or more o f sperm may fail to survive the process (Leidl et ah, 1993) and freezing o f bul l sperm remains a relatively inefficient procedure. A s penetration o f the sperm head through the zona pellucida requires vigorous propulsion (hyperactive motility) by the sperm tail, having a motile population of sperm is of great importance. The swim-up technique has become a well-accepted step in obtaining a highly motile population o f sperm for use in cattle I V F (Lathrop and Foote, 1986). In practice, however, there may be instances in which this procedure is omitted prior to washing the sperm. The need to use the swim-up may be greater when semen quality and sperm motility are poor (Ing et al, 1991; Catt, 1990). The washing of bull sperm in order to remove seminal plasma 35 prior to its use in I V F is wel l recognized as to be an essential step (Lu et al, 1987; L u 1990). Washing the bul l spermatozoa by centrifugal sedimentation and resuspention in fresh medium is generally agreed to be the most rapid and effective method of removing seminal plasma. In the sperm preparation method employed in cattle I V F in Dubl in (Lu et al, 1987; L u 1990), motile sperm cells, recovered after swim-up into T A L P medium (Ijaz and Hunter, 1989) are centrifuged twice at 500 g for 10 minutes. Caffeine, a cyclic nucleotide phosphodiesterase inhibitor, has been employed as an agent to stimulate the motility o f bul l sperm; it is probably effective by inhibiting phosphodiesterase, which results in an intracellular accumulation o f second messenger, c A M P . The effect o f caffeine on human sperm motility has been widely documented (Garbers et al, 1971; Traub et al, 1982; Ba l l and First, 1983; Crister et al, 1984; El-Gaafary et al, 1990). In cattle IVF , caffeine treatment o f sperm has been reported to result in increasing penetration rates (Niwa et al, 1988). Matured oocytes are inseminated with washed sperm shortly after preparation (Chen et al, 1992). In cattle IVF , the commonly used sperm doses are 0.5 to 5 mi l l ion sperm/ml. Expressed in terms of sperm:oocyte ratios, these would vary from 10,000 to 20,000: l)(Brackett et al, 1982; Bousquet et al, 1984; Lambert et al, 1986; Parrish et al, 1986b; L u et al, 1987b). However, in some reports, the ratio has been reduced to as low as 500:1 (First and Parrish, 1987a, 1987b). After sperm preparation, the oocyte^ should be prepared for fertilization. Sirard and Lambert (1986) stressed that the quality o f oocytes recovered from cattle is important for the success of IVF . According to these authors, oocyte should present an ideal morphology, which means showing an expanded and clear cumulus cell mass around the oocyte. This characteristic o f oocytes ensured that 50% cleavage rate after I V F ; where the recovered oocyte showed a compact corona radiata layer o f cells, no more than 6% could be expected to cleave. 36 It was concluded that the variability in cattle I V F results might be attributable to defects in either oocyte quality or sperm quality (Lambert et al, 1986). Several different methods have been used to remove some or all o f the cumulus cell layers, in order to make the surface of the in virro-matured cattle oocyte more accessible. Many different methods for this purpose have been tried such as, mechanical stripping in suitably sized micropipettes (Lu et al, 1987b, 1988a; Cox, 1991), use of hyaluronidase preparations (Xu et al, 1987; Park et al, 1989), and vortexing (Parrish et al, 1988). Younis and Brackett (1991) reported that cumulus cells are necessary at the time of I V F to maximize the incidence of A R . A s early as the report o f Ba l l et al (1983), the presence of some cumulus cells around the oocyte was recorded as beneficial. Mature bovine oocyte has a very limited life span in vivo. Similarly, in the cattle I V F system sperm should meet with the oocyte shortly after its maturation and under the appropriate temperature conditions. It has been reported that secondary oocytes are very sensitive to reductions in temperature (Moor and Crosby, 1985; Pickering et al, 1990). For such reasons, there should be careful control o f the temperature environment o f the secondary oocyte. There are some reports that, in vivo, the oocyte maturation period is about 24 hours in the cow (Lu et al, 1987a, 1987b, 1988a, 1988b, 1988c). Susko-Parrish et al (1991) examined the influence of aging in cattle oocytes produced by an in vitro maturation system, the time at which sperm penetrated the oocytes was not affected by the age factor, but there was evidence of reduced embryonic development in oocytes aged 32 hours. There is some flexibil ity in the duration o f maturation periods as no significant differences in cleavage rate were found between 18- and 24-hour in vitro maturation periods, but blastocyst yield was higher at the 24-hour period (Monaghan et al, 1993). Unt i l now, many factors have been recognized as contributing towards the successful interaction o f oocyte and sperm in cattle IVF . Various possibilities exist in terms of the culture systems for cattle IVF . In Dubl in, the procedure employed by L u et al. (1987b, 1988a) is based 37 on using micro-droplets o f modified T A L P (50 ul) on a plate under mineral o i l . This overlying oi l serves both to minimize evaporation and to prevent the entry o f microorganisms. The T A L P medium is a modified Tyrode's preparation (Bavister and Yanaagimachi, 1977) containing 25 m M sodium bicarbonate and B S A (0.6%). In preparing media, glassware should preferably be silicone-coated to prevent adsorption or contamination problems. Water quality in the I V F system must be closely monitored and carefully controlled. It has been reported that fertilization o f cattle oocytes is a temperature-dependent process (Lenz et al, 1983a). A temperature o f 38-39°C has been reported to be optimal for both bovine oocyte maturation and sperm penetration (Lenz et al., 1983a; Iqbal and Hunter, 1992). It is important to incubate gametes and embryo in a rigorously controlled atmosphere of either 5% carbon dioxide in air or 5% carbon dioxide, 5% oxygen and 90% nitrogen, using maximum humidity to prevent medium evaporation. Workers in cattle I V F have exposed oocytes to sperm individually or in groups of varying numbers (e.g. 5-20). It is generally believed that I V F may be more effective when oocytes are exposed to sperm in groups rather than singly (Hensleigh and Hunter, 1983). The implication is that oocytes and associated cells may produce or secrete substances promoting fertilization. In the live cow, it is believed that fertilization occurs in the presence of few sperm in the fallopian tube, but these sperm would be in contact with oviduct cells, as in the case of ovulated oocyte. There may be substances produced by oviductal cells which are especially important for capacitation o f sperm (Pollard et ah, 1991b) as wel l as for development o f the embryo after fertilization. In conclusion, the efficiency of I V F procedures depends on appropriate quality control and there are several excellent reviews dealing with these (Boone and shapiro, 1990; Schiewe etal, 1990). 38 CHAPTER 3 EFFECTS OF 17-B ESTRADIOL, PROGESTERONE, AND CHOLESTEROL ON SPERM ACROSOME REACTION AND MECHANISMS OF CHOLESTEROL EFFECTS Progesterone (P4) is known to induce mammalian sperm acrosome reaction (AR) in vitro, whereas cholesterol (CH), one of the major components in seminal plasma, inhibits A R . The objectives o f this study were to elucidate the in vitro effects of P4, 17 p-estradiol (E2), and C H on A R of cryopreserved bovine sperm as wel l as to study the cellular mechanisms of C H effects on P4-induced A R . Swim-up separated sperm from six bulls were incubated in modified Tyrodes' medium for 0, 2, 4, and 6 hr at 38.5°C in a 5% CO2 incubator. Mot i l i ty and viability o f sperm were estimated after thawing and after each incubation time. Acrosome reaction was induced with P4 (10 pg/ml), dibutyryl adenosine 3' :5'-cycl ic monophosphate (db-cAMP) (1 mM) , forskolin (10 uM) , and Ca 2 +- ionophore (10 u.M), either in the presence or absence of C H (1 p.g/ml). The effects o f P-sitosterol (1 u,g/ml), a C H plant analog, and C H on P 4- induced A R were also compared. Percentages of sperm A R were determined using fluorescein-labeled lectin, from pisum sativum as the probe. In addition, P 4 - and db-cAMP-induced A R was assessed for correlation with the in vivo fertility rates. Acrosome reaction was induced by all inducers immediately after thawing sperm and optimal A R induction was achieved by adding P 4 , d b - c A M P , forskolin, and Ca 2 +- ionophore for 30 min incubations (34.4 ± 2.5%; 49.3 ± 2.0%; 81.8 + 1.2%; 95.7 ± 1.0%; respectively). The extents of A R induction by P 4 , d b - c A M P , forskolin, and Ca 2 +- ionophore did not differ at 0, 2, 4, and 6 hr of incubation. Cholesterol and P-sitosterol (95.6% and 95.4% inhibition) inhibited P 4- induced A R in an identical manner. 39 Dibutyry l -cAMP-induced A R was also inhibited by C H but this was not the case for C a -ionophore. Forskolin-induced A R was partially inhibited by C H (21.2%). The results of the current study seem to suggest that frozen-thawed bovine sperm are fully capacitated at thawing. P4, d b - c A M P , forskolin, and Ca 2 +- ionophore induce A R in frozen-thawed bovine sperm effectively with some degree of individual variation. In contrast, E 2 has little effect on A R . It seems that no relationship was found between P4-, db -cAMP- , forskolin-, and Ca 2 +- ionophore induced A R and in vivo fertility o f the bulls. Cholesterol strongly inhibits A R induced by P4 as wel l as by db -cAMP. It appears that C H probably inhibits A R in a non-competitive manner by modifying the structure o f the sperm plasma membrane, thereby, masking the surface receptors for P4 binding. 3.1. I N T R O D U C T I O N Mammalian sperm are not immediately capable of fertilizing oocytes, rather they should undergo a series o f preparational alterations which normally take place in the female reproductive tract (Austin, 1951; Chang, 1951). Two stages of changes occur in sperm which involve an initial sperm membrane alteration (Yanagimachi, 1981; Langlais and Roberts, 1985) and subsequently the fusion of the plasma membrane and outer acrosomal membrane (Austin and Bishop, 1958; Barros et al, 1967; Yanagimachi and Usu i , 1974; Russel et al, 1979; Meize l , 1984; Lanlais and Roberts, 1985; Yud in et al, 1988; Gadella et al, 1995). The first stage is called capacitation, which includes a series of reversible subcellular changes eventually allowing the influx of C a 2 + . Capacitation was first described by Chang (1955) and Austin (1951,1952) and reviewed by several researchers (Flormann and Babcock, 1991; Yanagimachi, 1994; De Lamirande et al, 1997; Cross, 1998; Florman et al, 1998 ). The second phase is termed A R which is one of the most important sperm physiological functions during the initial fertilization processes in vitro and in vivo (Yanagimachi, 1981,1988). 4 0 The process of capacitation, first reported over four decades ago is stil l not very wel l understood. The physiological site o f capacitation in vivo is the oviduct or the uterus depending on species (Yanagimachi, 1994). The factors in the female reproductive tract responsible for capacitation and A R , are still unknown (Bedford, 1983; M c Nutt and K i l l ian , 1991). Oviduct f luid comprises both a secretive transudate o f serum and a secretary product from the oviduct epithelium (Oliphant et al, 1978). Oviduct secretions affect sperm capacitation and motility in vivo and in vitro in different species, hamster (Yanagimachi, 1969), rabbit (Chang and hunter, 1972), mouse (Chang and Hunter, 1972), and bull (Parrish and Susko-Parrish, 1989). A t ovulation, follicular fluid is transported to the oviduct (Sliwa, 1993). The ability o f this fluid to capacitate (Gwatkin and Anderson, 1969; Yanagimachi, 1969) and initiate A R (Lui et al, 1977; Tadano and Yamada, 1978; Tesarik, 1985; Suarez, 1986; Bryant et al, 1988; Parrish and Susko-Parrish, 1989; M c Nutt and K i l l ian , 1991; Hansen et al, 1991) o f various mammals spermatozoa has been wel l established. Substances that induce A R in vivo (only in capacitated sperm) (Parrish et al, 1988), may be the zona pellucida as described for mice (Florman et al, 1994) and for human (Meizel , 1985; Wassarman, 1987; Cross, 1988) or other components o f the cumulus/oocyte complex (Siiteri et al, 1988; Stock et al, 1989). Progesterone has been strongly reported in the fluid of preovulatory human ovarian follicles as an active factor involved in A R induction (Osman et al, 1989). It has also been reported as a steroid secreted by the cumulus oophorus/follicular cells which remain associated with the ovulated human oocytes, in vivo (Sabeur et al, 1996). Since the cumulus is present at the time of fertilization, progesterone should also be present and may play a role in initiating the A R of fertilizing sperm in vivo (Kopf and Gerton, 1991; Yanagimachi, 1994). Indeed the A R timing is ensured by the action o f physiological inducers present in the zona pellucida (Cross et al, 41 1988), in the cumulus oophorus (Tesarik, 1985; Teasrik et al, 1988; Siiteri et al, 1988), and in the follicular f luid (Tesarik, 1985; Suarez et al, 1986; Thomas and Meize l , 1988). Since 1989, a number of studies have demonstrated the effects o f progesterone on human sperm functions, particularly the facilitating effect of progesterone on intracellular calcium influx leading to A R (Lee et al, 1998; Yud in et al, 1988; Parinaud et al, 1992; Sabeur and Me ize l , 1995; Doherty et al, 1995; Blackmore et al, 1990). However, the precise role o f progesterone on the signal transduction pathways involved in A R is stil l incompletely understood although interactions between progesterone and its receptors on the sperm surface have been implicated (White and Aitken, 1989; De Jonge et al, 1991; Rotem et al, 1992; Furuya et al, 1993; Biel feld et al, 1994). Recent studies suggest that progesterone-induced A R involves both an early and a latent calcium influx in human spermatozoa by triggering the opening o f a plasma membrane calcium channel and activating protein-tyrosine kinase (PTK) respectively (Tesarik et al, 1996). Further, these two distinct reactions might involve different progesterone receptors (Blackmore et al, 1991; Sabeur et al, 1996; Tesarik et al, 1996). During the transit from male to female reproductive tracts, the spermatozoa is exposed to a variety o f steroids including testosterone, estradiol and progesterone. Since these steroids are hydrophobic in nature, they would be expected to bind to spermatozoa and elicit physiological effects. But this does not seem the case in human, and only progesterone appears to induce the acrosome reaction and the influx o f calcium ions into sperm, but not the other steroids (Blackmore et al, 1990). In the present study, experiments were performed to see i f progesterone and 17-0 estradiol could induce A R in cryopreserved bovine sperm similar to those in human sperm. It is expected that information obtained from this study w i l l further advance our knowledge regarding the effects of these steroids on mammalian sperm A R . 42 Another objective o f this study was to elucidate the effects of exogenous cholesterol on the spontaneous and progesterone-induced A R in cryopreserved bovine sperm. Sterols are structural lipids present in the membranes of most eukaryotic cells. The characteristic structure of sterols is the steroid nucleus including four fused rings, three with six carbons and one with five (Lehninger et al, 1993; Appendix E) . Since the fused rings do not allow rotation about C -C bonds, the steroid nucleus is almost planar, and relatively rigid. Cholesterol (27 C) , the major sterol in animal tissues, is amphipathic in nature, with a polar head group (the hydroxyl group at C-3) and a non-polar hydrocarbon body (16-C fatty acid) (Lehninger et al, 1993; Appendix E) . The role of cholesterol in sperm fertilizing ability was first suggested by Davis, who found that cholesterol-containing vesicles from rabbit seminal plasma prevented capacitated sperm from fertilizing eggs in vivo (Davis, 1978). Cholesterol may also inhibit fertilization in vitro (Davis, 1976a; Go and Wolf, 1985) however, two other studies failed to confirm this effect (Bleau et al, 1975; Fyarer-Hosken et al., 1987). Among different type of cells, sperm plasma membrane has a unique l ipid composition, which is different from that of somatic cells (Poulos and White, 1973; Mack et al, 1986; Alvarez and Storey, 1995). Among these l ipid components, cholesterol plays a major regulatory role in the fluidity o f the l ipid bilayers such as membrane permeability, the lateral mobil i ty o f integral proteins, and functional receptors within the membranes (Davis, 1978; Langlais and Roberts, 1985; Benoff, 1993a). Several lines of evidence indicate that sperm capacitation involves a decrease in the membrane cholesterol/phospholipid ratio (Langlais and Roberts, 1985). A lbumin (Davis et al, 1979, 1980; Go and Wolf , 1985; Benof et al, 1993a, 1993b) and high-density lipoprotein (HDL) (Langlais et al, 1988; Ehrenwald et al, 1990) present in the female genital tract facilitate the efflux of cholesterol during the early phase o f capacitation. H igh density lipoprotein, has been reported to be the only class of lipoprotein in bovine follicular and oviductal fluids which may be responsible in part for cholesterol removal 43 from sperm plasma membrane (Brantmeier et al, 1987; Ehrenwald et al, 1990; Jaspard et al, 1996). Cholesterol is also known to be one of the major components of seminal plasma which inhibits A R , both in human and bovine sperm (Langlais and Roberts, 1985; Ehrenwald et al, 1988a, 1988b; Sugkraroek et al, 1991; Benoff et al, 1993b; Zarintash and Cross, 1996). Human sperm become responsive to inducers of the A R only i f they are washed free of seminal plasma (Yanagimachi, 1980). Our knowledge about the role of cholesterol in the induced A R is stil l far from complete. Addit ion o f exogenous cholesterol to capacitation medium prevented fresh human sperm from becoming acrosome-reacted either spontaneously (Davis, 1980; Fleming and Yanagimachi, 1981; Brantmeier et al, 1987; Jaspard et al, 1996; Motamed Khorasani et al, in press) or by using A R inducers (Cross, 1994; Zarintash and Cross, 1996; Motamed Khorasani et al, in press). The inhibitory effects of cholesterol on the progesterone surface receptors have been originally studied by Motamed Khorasani et al. (in press). However, the underlying mechanisms involved, in particular the mechanism of its inhibitory effect on progesterone-induced A R remain unknown. One potential mechanism is that the presence of cholesterol may modify the structure or fluidity of sperm plasma membrane, thereby, exposing progesterone surface receptors for ligand binding and action. In this study, experiments were performed to further elucidate the cellular mechanisms of cholesterol effects on progesterone-induced A R in bovine sperm. Despite the availability of standard techniques to assess important semen characteristics such as motility, viability, morphology, freezability, and acrosomal integrity, in vitro, no reliable parameter has been established to predict fertility (Salisbury et al, 1978). It is unlikely that a simple test or even a series of tests can be used toward accurate evaluation o f semen (Saacke, 1970), yet it is important to continually look for techniques that may identify new 44 components or characteristics of semen which influence fertility. A s conventional parameters o f semen analysis have shown their limits in predicting male fertility potential, the study o f more delicate aspects o f human sperm, such as A R appears quite promising. In this thesis, experiments were performed to elucidate the correlation between progesterone-, db - cAMP- , forskolin, and Ca 2 +-ionophor-induced A R of cryo-preserved bovine sperm and in vivo fertility (based on 90-day nonreturn rates). Establishment o f a good correlation probaly w i l l lead to development o f a laboratory test to predict the performance of bulls in the field, which would provide both livestock breeders and A I organizations with significant economic gains derived form improved breeding efficiency (Saacke, 1970; Salisbury et al., 1978; Saacke, 1982; Hafez, 1987). 3.2. M A T E R I A L S A N D M E T H O D S 3.2.1. Chemicals The fol lowing items were purchased from Sigma chemical company, St. Louis, M O , U S A : bovine serum albumin (BSA) , fluorescein isothiocyanate-labeled pisum sativum agglutinin (F ITC-PSA) , progesterone P-cyclodextrin complex (water soluble progesterone), cholesterol p-cyclodextrin complex (water soluble cholesterol), P-cyclodextrin, 17 p-estradiol, heparin sodium salt (heparin sulfate), forskolin, sodium bicarbonate, sodium chloride, potassium chloride, sodium phosphate monobasic, sodium lactate, calcium chloride, magnesium chloride, H E P E S (1M), sodium pyruvate, Gentamicin, 2'-o-dibutyryl adenosine 3':5'-cyclic monophosphate (db-cAMP), calcium ionophore A23187, p-sitosterol, dimethyl sulfoxide (DMSO) . In this study, progesterone- and cholesterol-P-cyclodextrin complexes were used mainly to make progesterone and cholesterol, which are hydrophobic in nature, water-soluble, and to 45 decrease the possible toxic effects o f D M S O on sperm. The exterior o f the P-cyclodextrin molecule is extremely hydrophilic which allows an interaction with water while its apolar, hydrophobic cavity is the result o f the non-bonding electron pairs o f the glycosidic oxygen "bridges" being oriented toward the center of cavity. This cavity allows complexation with hydrophobic molecules (Bender and Komiyama, 1978). The chemical structure o f P-cyclodextrin, progesterone, cholesterol, 17 p-estradiol, and p-sitosterol are presented in Appendix E. 3.2.2. Heparin preparation Heparin as a sodium salt (170 units/mg) was dissolved in saline as a 100 x stock. Via ls o f heparin stock were stored frozen at -20°C until used. 3.2.3. Culture media Modi f ied Tyrode's medium (TALP) was used for experiments discussed in this chapter (Bavister and Yanagimachi, 1977; Parrish et al, 1988). Modifications of T A L P used for sperm cultures ( S P - T A L P ) and T A L P used for sperm capacitation ( H - T A L P ) (Handrow et al, 1982; Parrish et al, 1985a, 1985b, 1985c, 1986,1988) are presented in Appendix B. Glucose was not added in S P - T A L P and H - T A L P , because it inhibits the effects o f heparin on bovine sperm capacitation (Parrish et al, 1985a, 1985b, 1985c; Susko-Parrish and Parrish, 1988; Leibfried-Rutledget et al, 1989; Handrow et al, 1989). Energy substrates were present in the form of lactates and pyruvates in the media. A touch o f phenol red was used as a p H indicator in the media (in p H 6.8, yellow and in p H 8.2, red). A l l media were filtered through 0.22-um filters before use. 46 3.2.4. Semen samples Semen samples from six Holstein bulls were used in this study (#444, #389, #461, #391, #476, #436). Three different ejaculates from each bul l were used with a total of 18 experimental replicates for each treatment. Cryopreserved semen samples were provided by Westgen, Mi lner, Bri t ish Columbia, Canada. 3.2.5. Sperm preparation The cryopreserved semen samples from each bul l were from one collection, split and frozen in 0.5-ml straws. Frozen straws were thawed in a 37°C water bath for 40 seconds. Spermatozoa were then washed twice with modified Tyrode's medium ( S P - T A L P medium, p H 7.4, Appendix B ) (De Jonge et al., 1989).at 800 x g for 3 minutes. The washed pellet o f sperm was then covered with 1-2 ml of S p - T A L P medium for a 60 minute swim-up (38.5°C and 5% CO2, 100% R H ) to obtain the most motile population of spermatozoa as described previously (Biggers et al., 1971; Lopata et al., 1976; Overstreet etal., 1980; Wright and Bondio l i , 1981; Lenz et al., 1983b; Parrish et al., 1986; Wang et al., 1991). Swim-up separated sperm in modified T A L P medium were centrifugated at 500 x g for 5 minutes (Lee et al., 1987; Margalioth et al., 1988; Okabe et al., 1992). The sperm pellet was resuspended in known amounts o f Tyrode's medium supplemented with 10 |ag/ml heparin sulfate ( H - T A L P ; Appendix B ) (Handrow et al., 1982; Parrish et al., 1985a, 1985b, 1986, 1988). The sperm concentration was determined by using the Neubauer hemocytometer method (World Health Organization, 1992). 47 3.2.6. Motility and viability assessment Quantitative motility was determined by counting both motile and non-motile spermatozoa in at least 10 separate and randomly selected fields (but not near the coverslips edge). A t least 100 spermatozoa were counted. The percentage o f motile spermatozoa was calculated from the mean percentage motility for all fields counted (World Health Organization, 1992). Moti l i ty and viability of the sperm were estimated after thawing and after each capacitation incubation time. The sperm viability was determined by util izing the supravital staining technique, involved staining the spermatozoa first with 1% eosin in distilled water and subsequently counterstaining with 10% nigrosin in distilled water. The slides were observed under the ordinary light microscope. One hundred spermatozoa were counted differentiating the dead from the live. Under the light microscope, the dead spermatozoa appeared red and the live ones were unstained (colorless) (World Health Organization, 1992). 3.2.7. Capacitation procedure Fol lowing swim-up, the sperm concentration was estimated by hemocytometer and adjusted to 5 x 10 6 sperm/ml (Biggers et al, 1971; Overstreet et al, 1980; Blackmore et al, 1990; Blackmore and Lattanozio, 1991; Falsetti et al, 1993). Sperm specimens were incubated in 100 u,l drops of capacitation medium ( H - T A L P ; Appendix B ) under mineral o i l and in a 96-wel l culture dish for 0 , 2 , 4 , and 6 hours for capacitation (38.5°C and 5% CO2 in humidified air, 100% relative humidity). 48 3.2.8. Direct immunofluorescence assay After each treatment, a 5 u.1 droplet o f sperm solution, was placed on teflon-coated multi-spot slides (Fisher Scientific Ltd., Ottawa, O N , Canada), spread uniformly to cover the wel l , air-dried, and methanol-fixed. Each wel l was washed three times with P B S and 0.5% B S A . Fluorescein isothiocyanate-labeled pisum sativum agglutinin (F ITC-PSA; Sigma, St. Louis, M O , U S A ) was used as a probe for the assessment of acrosomal status. P S A is a plant lectin known to bind to intra-acrosomal components (Cross et al., 1986; Mendoza et al, 1992; Cross and Watson, 1994). The acrosomal region of acrosome-intact spermatozoa shows a bright fluorescent staining with F I T C - P S A , whereas acrosome-reacted spermatozoa show no staining (Cross and Meize l , 1989) (Plate 3.1.). A 2 mg/ml solution o f F I T C - P S A was diluted 1:100 and a twenty five ul droplet of that applied to the slides (final concentration o f 20 u,g/ml). The usefulness of fluorescein-labeled lectins, including P S A , for assessing acrosome status in bul l spermatozoa has been recently described by Cross and Watson (1994). After incubation of sperm with fluorescent dye for 30 minutes in dark chamber and 38.5°C, excess P S A was removed and slides were washed three times with P B S . A droplet of 80% glycerol in Dulbecco's phosphate buffer saline (PBS) was placed on each wel l and the slides were mounted by coverslips. Coded slides were examined at 400 x to 1000 x magnification with a microscope equipped with epifluorescence optics (excitation filter range: 450-490 nm; barrier filter 520 nm) and photographed on 35 mm/400 A S A f i lm for color slides (Kodak Canda Inc., Toronto, ON) . Sperm counts were made on randomly selected fields until 200 total spermatozoa were counted from each wel l o f the slides. In each field, the number o f total spermatozoa was initially counted under bright field il lumination, immediately followed by the counting of FITC-labeled sperm under fluorescent light in dark field to obtain the percent acrosome-intact sperm. Only the sperm with a complete, uniform fluorescent staining of 49 acrosomal region were scored as acrosome-intact, while spermatozoa showing irregular or patchy fluorescence, those showing band-like fluorescence pattern on the equatorial segment, and those with no fluorescence were considered as acrosome-reacted. A step by step protocol for direct immunofluorescent assay is presented in Appendix F. Live sperm under the light microscope Al AR AR PLATE 3.1. Schematic illustration of acrosome-intact and acrosome-react spermatozoa stained with PSA-FITC. 3.2.9. Exper imenta l designs This study incorporated three objectives: 1) assessment o f capacitation status in cryo-preserved bovine sperm, 2) the effects of progesterone, 17 P-estradiol, and cholesterol on cryopreserved bovine sperm, 3) the cellular mechanisms o f cholesterol effects on bovine sperm. To increase the efficiency and accuracy o f the experiments all 16 treatments were carried out in one experiment in order to further elucidate the above objectives. A list o f treatments is presented in Table 3.1. 50 T A B L E 3.1. Treatments carried out in Chapter 3 Treatment Number Treatments* Concentration 1 Control 2 P-Cyclodextrin§ lOug/ml 3 Progesterone lOug/ml 4 Db-cAMP 1 mM 5 Ca2+ Ionophore 10 uM 6 Cholesterol 1 ug/ml 7 Cholesterol + Progesterone 1 ug/ml + lOug/ml 8 Cholesterol + Db-cAMP lug/ml + 1 mM 9 Cholesterol + Ca2+ Ionophore lug/ml + 10 uM 10 p-Sitosterol lug/ml 11 P-Sitosterol + Progesterone lug/ml + lOug/ml 12 DMSO^ < 1% final concentration in medium 13 17 P-Estradiol lOug/ml 14 Ethanol''' < 1% final concentration in medium 1 5 Forskolin 1 0 u M 1 6 Cholesterol + Forskolin lug/ml + 10 uM s . Served as negative control for progesterone and cholesterol. \ Served as negative control for Ca++ Ionophore and P-Sitosterol. Served as negative control for 17 P-Estradiol. . All of these treatments were carried out in four different incubation times (0. 2,4, and 6 hours). 3.2.9.1. Pre l im inary studies A preliminary study with three different concentrations of progesterone (0.1, 1, and 10 p-g/ml) (Blackmore et al., 1994; Sabeur et al., 1996; Lee et al., 1998), 17 P-estradiol (0.1, 1, and 10 p.g/ml) (Thomas and Meize l , 1989; Blackmore et al., 1990; Meize l et al., 1990), and cholesterol (0.1, 1, and 10 p.g/ml) (Zarintash and Cross, 1996; Cross, 1996; Lee et al., 1998) were carried out to find the optimum concentration o f these steroids (Appendix H) . 51 3.2.9.2. Capacitation status of cryo-preserved bovine sperm 3.2.9.2.1. Spontaneous and progesterone-induced A R (Treatments #1-3 from Table 3.1.) To assess the sperm capacitation status at each incubation time, both spontaneous A R and progesterone-induced A R assays were performed. The percentages of spontaneous A R (percentage A R increasing with sperm incubation in capacitation medium as a function of time) were determined both in the whole model and for each individual bul l . Progesterone-P-cyclodextrin was diluted with H - T A L P , distributed in 200-ul plastic vials, and stored in -20°C until use. Based on the results o f the preliminary study, progesterone concentration o f 10 u,g/ml was used for all experiments. In order to study the capacitation status o f the sperm, progesterone was util ized as an A R inducer. A s a general control (treatment #1), swim-up separated sperm were incubated in capacitation medium (H -TALP) at 38.5°C, 5% CO2 in air, and maximum humidity for 0, 2, 4, and 6 hour intervals. In treatment #2, at each time interval, progesterone-p-cyclodextrin complex was added to sperm preparations for 30 minutes (Cross, 1993; Lee et al., 1998) to induce A R . In treatment #3, as a parallel negative control for progesterone, p-cyclodextrin of comparable concentration was added to the sperm preparations, at similar incubation conditions. The acrosomal status was assessed .and the percentages of A R induced by progesterone were determined both in the whole model and for each individual bul l . 3.2.9.2.2. Variation of motility and viability during capacitation In order to study the effects of incubation of frozen-thawed spermatozoa in capacitation medium, motility and viability were estimated after thawing and after different incubation times for all samples using W H O criteria as mentioned previously. The percentages o f decrease in motility and viability over 2, 4, and 6 hours of capacitation incubation were 52 determined. The correlation between the percentages of post-thaw motility and the in vivo fertility of bulls was determined. The relationship between the percentages of motility decrease over the 2-hour incubation period and the in vivo fertility of bulls (non-return rates) were also studied. 3.2.9.3. The effects of progesterone, 17 P-estradiol, and cholesterol on bovine sperm A R (Treatments #6,7,13 and 14 from Table 3.1.) Assessment o f the effects of progesterone has been discussed in section 3.2.9.2.1. In order to assess the effects o f 17 P-estradiol and cholesterol, several treatments were carried out. 17 P-estradiol was first dissolved in ethanol (Shivaji et al., 1992), and then diluted with H -T A L P and distributed in 200 u l plastic vials and stored at -20°C until use. The final concentration o f ethanol in the dilution was set to less than 1% to minimize the effect o f solvent on the experiments. Based on the results o f the preliminary studies, 17-p estradiol at 10 ug/ml and cholesterol at 1 p.g/ml was used for all treatments. Two different treatments were carried out in case of 17-P estradiol. In treatment #13, 17 P-estradiol was added to sperm preparations for 30 minutes, at each time interval, to induce A R . A s a negative control for 17 p-estradiol (treatment #14), Ethanol (1% solution in H - T A L P medium) was added to the sperm preparations, at each incubation time, for the same incubation period as mentioned previosly. Two treatments were also carried out to study the effects of cholesterol. In treatments #6, cholesterol-P-cyclodextrin complex was added to sperm preparations for 30 minutes (Graham and Foote, 1987a, 1987b; Wi lhelm et al., 1996; Lee et al., 1998), at each time interval, as previously described (Lee et al, 1998). In treatment #7, capacitated sperm for different times were incubated with cholesterol for 10-15 minutes (Graham and Foote, 1987a, 1987b; Cross et al, 1993; Wi lhelm et al, 1996; Lee et al, 1998) 53 followed by progesterone addition to the sperm solution for a 30 minute-incubation. Final ly, the sperm were methanol-fixed on slides for acrosomal status assessment. The effect of 17 P-estradiol and cholesterol on A R changes in bul l sperm was determined, both in general and for each individual bul l . 3.2.9.4. The mechanisms of cholesterol effects on bovine sperm A R 3.2.9.4.1. Comparison of cholesterol and P-sitosterol effects on progesterone-induced A R (Treatments #10,11,12 from Table 3.1.) In order to study the specificity of the cholesterol effects, P-sitosterol which is a general plant sterol (Vander kraak and Mac Latchy, 1995; Fernandes et al, 1996; Nagy et al, 1996; Mac Latchy, 1997; Nguyen et al, 1999; Zakaria et al, 1999), was used in this study. Previously capacitated sperm were incubated with P-sitosterol (1 p-g/ml) in different time intervals (treatment #10). D M S O (diluent for P-sitosterol with final concentration o f less than 1% in sperm solution) was used as a negative control for P-sitosterol (treatment #12). Capacitated sperm were incubated with p-sitosterol in different time intervals, progesterone (10 u,g/ml) was then added, 10 minutes after addition of p-sitosterol, and the incubation was continued for 30 minute (treatment #11). Fluorescein-labeled P S A was then used to assess acrosomal status. Results o f the experiments were compared for cholesterol and P-sitosterol effects on Progesterone-induced A R . 3.2.9.4.2. Effects of cholesterol on db-cAMP-, foskolin-, and calcium ionophore-induced A R (Treatment #4, 5, 8,9,15,16 from Table 3.1.) In order to elucidate the cholesterol effects on db-cAMP-induced A R , two treatments were carried out using db -cAMP, which is a more membrane permeable analogue for the 54 second messenger, c A M P (Bavister and Boatman, 1984). Capacitated sperm after different time intervals were incubated with db -cAMP (1 mM) (De Jonge et al, 1989; Doherty et al., 1995; Motamed Khorasani et al., in press), either with (treatment #8) or without (treatment #4) cholesterol (1 u.g/ml). Cholesterol was added first (De Jonge et al, 1989; Doherty et al, 1995), and 10-15 minutes later db -cAMP was added and the co-incubation continued for 30 minutes (De Jonge et al., 1989; Sofikitis et al., 1993). Fluorescein-labeled P S A was used in order to assess acrosomal status. Spermatozoa were counted from each treatment at each of the four time intervals. The levels of db-cAMP-induced A R were compared both among all individual bulls and in general. The percentage of cholesterol inhibition on db-cAMP-induced A R were also assessed in general. In order to elucidate the cholesterol effects on forskolin-induced A R , two treatments were carried out using forskolin. Capacitated sperm in different time intervals were incubated with forskolin (10 uM) (De Jonge et al, 1991; Doherty et al, 1995), either with (treatment #16) or without (treatment #15) cholesterol (1 p,g/ml). Cholesterol was added first and 10-15 minutes later forskolin was added and the co-incubation continued for 30 minutes (De Jonge et al, 1991; Doherty et al, 1995). In order to elucidate the effects o f cholesterol on calcium ionophore, capacitated sperm of different time intervals were incubated with calcium ionophore (10 uM) (De Jonge et al, 1989; L i u and Baker, 1990), either with (treatment #9) or without (treatment #5) cholesterol (1 p.g/ml). Cholesterol was added first (De Jonge et al, 1989), and 10-15 minutes later calcium ionophore was added and the co-incubation continued for 30 minutes (Aitken et al, 1984; De Jonge et al, 1989, 1991). In order to assess acrosomal status, spermatozoa were counted from each treatment at each of the four time intervals. The levels of calcium ionophore-induced A R 55 were compared both among all individual bulls and in general. The percentage o f cholesterol inhibition on calcium ionophore-induced A R was also assessed in general. 3.2.10. Field fertility data The in vivo fertility results o f all six bulls were obtained from the records o f the Brit ish Columbia Art i f ic ial Insemination Center. The degree o f correlation was estimated between in vivo fertility and A R inducibility of semen samples by progesterone, d b - c A M P , forskolin, C a 2 + -ionophore. Based on the results of the progesterone- and db-cAMP-induced A R , the total population studied were divided to two groups with higher and lower A R induciblity. Fie ld fertility data for each bull was based on the 90-day non-return rate to first insemination with semen o f that particular bul l . 3.2.11. Statistical analysis Statistical analysis employed analysis o f variance ( A N O V A ) uti l izing the J M P statistical package (SAS Institute Inc., Sas Campus Drive, Cary, North Carolina 27513, U S A ) . This experiment utilized a split-split plot design with the following statistical model: Y i j k l m = P- + B i + C(B)i j + I k + (BI)ik + E 2 + T l + (BT) i i + ( IT) k l + (BIT)ikl + E 3 (i = 1,2 6) G = 1,2,3) (k = 1,2,3,4) (1 = 1,2, 15) Where Y = Percent change in A R (Arcsine transformed); p, • population mean; B = effect of bulls; C = effect of ejaculates within bul l (this term is also used as the error term, E i , to test the bul l effect); I = effect of time intervals (0 ,2 ,4 , and 6 hours); T = effect o f treatment. (BI), (BT) and (IT) are the two-way interaction terms. (BIT) is the three-way interaction term. E2 is the error term used to test the effects involving intervals, I and (BI), while E3 is the error term for the model. In all experiments, results were reported as the mean values for each 56 set of data ± standard error o f the means (S.E.) and the level o f statistical significance was defined at a p value o f less than 0.05. 3.3. R E S U L T S 3.3.1. Capacitation status of cryo-preserved bovine sperm 3.3.1.1. Spontaneous and Progesterone-induced A R in cryo-preserved bovine sperm The percentages of sperm spontaneous A R were compared at different incubation times (0, 2, 4, and 6 hours) for all bulls (Table 3.2.). Percentages o f spontaneous A R was determined (the decrease in the percentages o f acrosome-intact sperm when sperm were incubated for 2, 4, and 6 hours compared to time zero). T A B L E 3.2. The average percentages o f acrosome-intact sperm from all bulls at 0 , 2 , 4 , and 6 hours o f incubation in capacitation medium (n = 72). capacitation Incubation Time (hour) 0 2 4 6 % Acrosome-intact Sperm^ 55.9±1.59 a 56.1±1.62 a 54.4± 1.573 54.4 ±1.58* . Values were reported as the mean value ± standard errors of the means. . Values with similar superscripts were not significantly different (p > 0.05). A s show in Table 3.2., percentages of acrosome-intact sperm showed no significant decrease after different incubation times (0 ,2 ,4 , and 6 hours). In fact, the results indicated that spontaneous A R did not take place during the 6 hours o f incubation time. The average percentages of acrosome-intact sperm at different incubation times, were also assessed and presented for individual bulls in Table 3.3. Progesterone -induced A R of all bulls were compared against different incubation times (0, 2, 4, and 6 hours). Progesterone-induced A R were 34.1 ± 1.6%, 34.6 ± 1.7%, 34.2 ± 2.0%, 57 and 34.8 ± 1.7% at 0, 2, 4, and 6 hours of incubation, respectively. The percentages o f progesterone-induced A R did not change significantly when sperm were incubated in capacitation medium for 2, 4, and 6 hours before progesterone addition. T A B L E 3.3. The average percentages o f acrosome-intact sperm in six individual bulls at 0, 2, 4, and 6 hours of incubation in capacitation medium (n = 72). Bull Number % Acrosome-intact Sperm t 0 hours 2 hours 4 hours 6 hours #476 62.6±3.2a 62.0±3.3a 60.6 ± 2.63 60.7 ± 3.03 #436 57.0±2.5b 58.2±2.8b 54.8±2.5b 55.8±2.8b #391 62.5±1.7a 62.9±1.7a 61.8 ±2.2a 60.7 ± 1.9a #389 55.8 ± 1.0b 56.1 ±0.9 b 55.1 ±0.9 b 54.7 ± 0.9b #444 48.3 ± 2.2C 47.7 ± 2.0° 46.6 ± 2.0C 46.7 ± 2.4° #461 49.1±2.5° 49.6 ± 2.8C 47.8 ± 2.2C 47.5 ± 2.4° t . Values were reported as the mean value ± standard errors of the means. Photographs o f progesterone effects on cryo-preserved bovine sperm both in light and fluorescent microscope are presented in Plate 3. 2. The percentages o f A R increase following progesterone treatment were also compared for all six individual bulls (Figure 3.1.). A s shown in Figure 3.1., there were differences (p < 0.05) among bulls in percent A R increase following progesterone treatment. Bu l l #444 (27.7 + 1.1%) showed the minimum and bul l #391 (43.2 ± 1.2%) showed the maximum extent of A R increase following progesterone treatment. Since there were no significant differences in the percentages of progesterone-induced A R at different incubation times, data from all four-time interval studies were combined. Percentages o f A R induced following progesterone treatment were then averaged for all bulls. Fol lowing progesterone treatment, the percentage of acrosome-reacted sperm increased 58 significantly by 34.4 ± 2.5% (p < 0.05, compared to p-cyclodextrin treatment in which the percentage o f acrosome-reacted sperm increased by 1.9 ± 0.4% (Figure 3.4.). F I G U R E 3.1. The percentages of progesterone-induced A R in six bulls (n = 72). xi U 50 45 40 35 30 25 20 15 10 5 0 32.19 C d 28.72' 0.8 3.4 43.15' 1.9 38.98 I ab 2.5 27.71 J_ 0.8 35.88 be 2.2 • Progesterone • B-cyclodextrin. #476 #436 #391 #389 #444 #461 Bu l l Numbers . Values were differences between acrosome reaction in control and after each treatment. . Values were reported as the mean value ± standard errors of the means. . Values with different alphabetical superscripts were statistically different (p < 0.05). 59 - * * • 4 • • A PLATE 3.2. P roges te rone- Induced A R . A ) U n d e r the f luorescen t m i c r o s c o p e . B ) T h e same field under the l ight m i c r o s c o p e . B o t h the s o l i d a r rows ( comp le te A R ) and the u n f i l l e d a r rows (patchy s ta in ing ) are cons i de r ed ac rosome-reac ted . 60 3.3.1.2. Decrease in sperm motility and viability during capacitation incubation The percentages of sperm motility were plotted against different capacitation incubation times (0, 2, 4, and 6 hours) for each individual bul l (Figure 3.2.). The post-thaw motility o f sperm showed a significant decrease (p < 0.05 compared to post-thaw motility) during the incubation in capacitation medium. Individual bull variations (p < 0.05) in percent post-thaw motility decrease were observed during incubation. Among the bulls, the major motility decrease took place during the first 2 hour of incubation (p < 0.05). After the first 2 hours o f incubation, no further statistically significant changes in motility were observed. The sperm from bul l #461 (23.3 ± 1.4%) had the least motility decrease over time. In contrast the sperm from bul l #436 (42.9 + 2.5%) had the greatest time-dependent motility decrease. The average time-dependent motility decreases for each individual bull are presented in F ig . 3.2. When the average sperm motility of all bulls were plotted against the capacitation time (Fig. 3.3.), there was a decrease of 26.7 ± 1.9 % in the sperm motility during the first two hours o f incubation in capacitation medium (p< 0.05) (Table 3.4.). Sperm motility remained at the same level afterwards. T A B L E 3.4. The average percentages o f sperm motility/viability decrease in all bulls during 6 hours o f incubation in capacitation medium (n = 18). Incubation time (hour) 2 4 6 stt % Motility Decrease in Capacitation Incubation 26.7 ± 1.9a 29.0 ± 2.0a 30.9 ± 2.0a §tt % Viability Decrease in Capacitation Incubation 1.01 ±0.28b 1.98±0.35b 2.88±0.36b . Values were the differences between motility in control and after each incubation time. . Values were reported as the mean value ± standard errors of the means. . Values with similar alphabetical superscripts were not statistically different (p > 0.05). 61 F I G U R E 3.2. The percentages of sperm motility decrease in six bulls during 2, 4, and 6 hours o f incubation in capacitation medium (n = 18). 0 T 1 T 2 4 6 Incubation Time (hour) (). Values in parentheses were the p values for the related group compared to control (P-cyclodextrin). N o significant correlation was found between the percentages o f motility decrease over 2-hour capacitation incubation and the in vivo fertility o f the bulls. Percentages o f post-thaw motil ity and the in vivo fertility of the bulls were not correlated. When the percentages of sperm viability were compared in different incubation time intervals (0, 2, 4, and 6 hours) for each individual bul l , the post-thaw viabil ity showed no significant decrease during the incubation from beginning of the incubation to 6 hours). The average viabil ity decreases from thawing to the time of incubation for each individual are presented in Table 3.5. 62 T A B L E 3.5. The average percentages o f sperm viability decrease in six bulls during 6 hours o f incubation in capacitation medium (n = 18). Bull Number P I % Viability Decrease in Capacitation Incubation Time 2 hours 4 hours 6 hours #461 1.00 ±0.50 2.67 ± 1.17 3.1710.93 #391 1.27±0.37 1.70±0..21 2.4310.81 #444 0.73 ±0.15 1.57 ±0.57 2.27 ± 0.22 #476 0.03 ± 0.03 1.57±0.26 2.03 ± 0.03 #389 2.33 ± 1.49 2.83 ±1.86 4.67 ±1.45 #436 0.67 ± 0.20 1.57 ±0.35 2.73 ± 0.72 . Values were differences between sperm viability in control and after each treatment. . Values were reported as the mean value ± standard errors of the means. F I G U R E 3.3. The percentages o f sperm motility and viability decrease in six bulls during 2, 4, and 6 hours o f incubation in capacitation medium (n = 18). • V i a b i l i t y • M o t i l i t y 0 2 4 6 Incubation Time (hr) . Values were reported as the mean value ± standard errors of the means. . Values with different alphabetical superscripts were statistically different (p < 0.05). 63 When the sperm viability changes o f all bulls were plotted against the capacitation time (Fig. 3.3. and Table 3.5.), there was no significant decrease in the sperm viabil i ty during the first six hours o f incubation. 3.3.2. The effects of progesterone, 17 p-estradiol, and cholesterol on bovine sperm A R Acrosome reaction were carried out by induction o f sperm with 17 P-estradiol for 30 minutes and the A R changes were compared with different incubation times (0, 2, 4, and 6 hours) for all bulls. The number of acrosome-reacted sperm were increased in 72 replications, when sperm were treated with 17 p-estradiol. Since there were no statistical differences in the percentages o f 17 P-estradiol-induced A R over different time intervals, data from al l four-time interval studies were combined. Percentage of A R increase following 17 P-estradiol treatment was then estimated for all bulls. Fol lowing 17 P-estradiol treatment, the percentage o f acrosome-reacted sperm increased by 4.5 ± 0.7% (p < 0.05, compared to ethanol as control treatment which increased the acrosome-reacted sperm by 0.8 ± 0.1 %). The percentages o f A R increase following 17 P-estradiol treatment were also compared for all six individual bulls (Table 3.6.). A s shown in Table 3.6., there were differences (p < 0.05) among bulls in percent A R increase following 17 P-estradiol treatment. Bu l l #461 (7.8 ± 0.3%) showed significantly higher changes of A R compared to other bulls 17 P-estradiol treatment. 64 F I G U R E 3.4. The percentages of A R increase following 17 P-estradiol treatment in six bulls (n = 72). 10 • 17 p-estradiol ++ 0 #476 #436 #391 #389 #444 #461 Bu l l Numbers § *. Values were differences between acrosome reaction in control and after each treatment. \ Values were reported as the mean value ± standard errors of the means. .^ Values with different alphabetical superscripts were statistically different (p < 0.05). A s previous results showed, no statistical differences were observed in the post-thaw sperm function over different capacitation incubation times. Based on this conclusion, data from all four time interval cholesterol studies were combined. Cholesterol was shown to completely inhibit progesterone-induced A R (Figure 3.5.) in bulls at the concentration o f 1 u.g/ml. Fol lowing progesterone treatment, the percentage of acrosome-reacted sperm increased by 34.4 ± 2.5% (p < 0.05, compared to P-cyclodexrin treatment in which the percentage o f acrosome-reacted sperm increased by 1.9 ± 0.4%). When progesterone was added to the sperm, pre-treated with cholesterol, the percentage o f acrosome-reacted sperm increased by 1.53 + 0.3%. Since no spontaneous A R was observed during the 6 hours o f sperm capacitation 65 incubation, the effects of cholesterol on spontaneous AR were not possible to be studied in cryopreserved sperm. In fact, when cholesterol alone was added to the sperm and incubated over time, no significant changes in percentages of acrosome-reacted sperm were observed, compared to control. 3.3.3. Mechanisms of cholesterol effects on progesterone-induced AR 3.3.3.1. Comparison of cholesterol and P-sitosterol effects on progesterone-induced AR After thawing, progesterone-induced AR of cryopreserved bovine sperm was independent of the incubation time from zero to six hours so the data from all four time interval studies were combined. At the progesterone concentration of 10 ug/ml, the percentages of acrosome-reacted sperm increased significantly by 34.4 ± 2.5% for all bulls (p < 0.05 compared to control treatment). In the control treatment with comparable concentrations of P-cyclodextrin, the percentages of acrosome-reacted sperm increased by only 1.92 ± 0.4% (Figure 3.5.). Co-incubation of sperm with 1 ug/ml of cholesterol and 10 ug/ml of progesterone resulted in a 95.6% reduction in the percentage of progesterone-induced AR (Figure 3.5.). Similarly, co-incubation of sperm with 1 p.g/ml of P-sitosterol and 10 u.g/ml of progesterone resulted in a 95.4% reduction in the percentage of progesterone-induced AR (Figure 3.5.). In fact, the inhibitory pattern of p-sitosterol on progesterone-induced AR was virtually identical to that of cholesterol (Figure 3.5.). 66 FIGURE 3.5. The effects of cholesterol and P-sitosterol on progesterone-induced A R of frozen-thawed bovine sperm (n = 72). 1. P-Cyclodextrin 2. DMSO 3. Progesterone (10 ug/ml) 4. Cholesterol (1 ug/ml) 5. P-Sitosterol (1 ug/ml) 6. Progesterone (10 ug/ml)+ Cholesterol (1 ug/ml) 7. Progesterone (10 ug/ml)+ P-Sitosterol (1 ug/ml) Sperm Treatments § . Values were differences between acrosome reaction in control and after each treatment. \ Values were reported as the mean value ± standard errors of the means. ^ Values with different alphabetical superscripts were statistically different (p < 0.05). 3.3.3.2. Cholesterol effects on db-cAMP-, forskolin-, and calcium ionophore-induced AR The mean percent changes of acrosome-reacted sperm increased significantly by db-cAMP in 72 replications. The percentages of db-cAMP-induced A R did not vary significantly with the incubation time from zero to six hours. The percentages of acrosome-reacted sperm were 48.6 ± 1.4%, 49.1 ± 1.6%, 49.5 ± 1.7%, and 49.8 ± 1.5% at 0, 2, 4, and 6 hours of incubation, respectively. Since there were no statistical differences in the percentages of db-cAMP-induced A R over different time intervals, data from all four-time interval studies were combined. Percentages of A R increase following db-cAMP treatment were then estimated for all bulls. At 67 the d b - c A M P concentration of 1 m M , the percentage of acrosome-reacted sperm increased by 49.26 ± 2.0% (p < 0.05 compared to control treatment) (Figure 3.6.). FIGURE 3.6. The effects of cholesterol on progesterone-, db -cAMP- , forskolin, and calcium ionophore-induced A R of frozen-thawed bovine sperm (n = 72). bfl Progesterone Db-cAMP Ca-Ionophore Forskolin Sperm Treatments • -Cholesterol • +Cholesterol Values were differences between acrosome reaction in control and after each treatment. . Values were reported as the mean value ± standard errors of the means. . Values with different alphabetical superscripts were statistically different (p < 0.05). (). Values in parentheses were the p values for the related group compared to control (P-cyclodextrin). The percentages of A R induction following db -cAMP treatment were also compared for all six individual bulls (Figure 3.7.). A s shown in Figure 3.7., there were differences (p < 0.05) among bulls in percent A R increase following db -cAMP treatment. The percentages of A R induction ranged from 45.14 ± 1.3%, 45.07 ± 1.8%, and 47.4 ± 1.1% for bulls #476,436, and 68 389, respectively to 57.23 ± 1.5% for bul l #391. Furthermore, co-incubation o f sperm with 1 p.g/ml of cholesterol and 1 m M of db -cAMP resulted in a 96.7% reduction in the percentage o f db-cAMP-induced A R (Figure 3.6.). Under the microscope, the appearance of acrosome-reacted spermatozoa in effect of db -cAMP was similar to that of progesterone-induced A R spermatozoa. FIGURE 3.7. The percentages of A R increase following db -cAMP treatment in six bulls (n = 72). • d b - c A M P #476 #436 #391 #389 #444 #461 Bu l l Numbers . Values were differences between acrosome reaction in control and after each treatment. \ Values were reported as the mean value ± standard errors of the means. ^ Values with different alphabetical superscripts were statistically different (p < 0.05). The mean percent changes o f acrosome-reacted sperm increased significantly by forskolin in 72 replications. The percentages of A R changes in effect o f forskolin-induced A R were 81.5 ± 0.8%, 81.1 ± 0.8%, 81.6 ± 1.1%, and 82.7 ± 0.8% at 0, 2, 4, and 6 hours of incubation, respectively. No statistical differences in the percentages o f forskolin-induced A R 69 were observed over different time intervals. Average percentages of A R increase following forskolin treatment were determined for six individual bulls (Figure 3.8.). A t the forskolin concentration o f 10 u M , the average percentage of acrosome-reacted sperm increased by 81.8 ± 1.2% (p < 0.05 compared to control treatment) (Figure 3.6.). F I G U R E 3.8. The percentages of A R increase following forskolin treatment in six bulls (n = 72). Forskolin #476 #436 #391 #389 #444 #461 Bu l l Numbers t . Values were differences between acrosome reaction in control and after each treatment. . Values were reported as the mean value ± standard errors of the means. K Values with different alphabetical superscripts were statistically different (p < 0.05). Furthermore, co-incubation of spermatozoa with 1 u.g/ml o f cholesterol and 10 u M of forskolin resulted in a 21.2% reduction in the percentage of A R changes showing a partial reduction compared to forskolin alone (Figure 3.6.). Under the microscope, the appearance of 70 acrosome-reacted spermatozoa in effect of forskolin was similar to that o f calcium ionophore-induced A R spermatozoa. The mean percent changes of acrosome-reacted sperm increased significantly by calcium ionophore in 72 replicate experiments. The percentages of A R changes in effect of calcium ionophore-induced A R were 95.5 ± 1.1%, 94.1 ± 2.1%, 96.0 ± 1.0%, and 95.9 ± 1.0% at 0, 2, 4, and 6 hours of incubation, respectively. Since there were no statistically meaningful differences in the percentages of calcium ionophore-induced A R over different time intervals, data from all four time interval studies were combined. Average percentages of A R increase fol lowing calcium ionophore treatment were determined for all bulls. A t the calcium ionophore concentration o f 10 u M , the percentage of acrosome-reacted sperm increased by 95.74 ± 1.0% (p < 0.05 compared to control treatment) (Figure 3.6.). When the percentages of A R increase following calcium ionophore treatment were compared for all six individual bulls, again there were differences (p < 0.05) among bulls in percent A R increase following calcium ionophore treatment (Figure 3.9.). Furthermore, co-incubation of sperm with 1 ug/ml o f cholesterol and 1 m M of calcium ionophore resulted in no reduction in the percentage of calcium ionophore-induced A R (Figure 3.6.). Photographs of calcium ionophore effects on cryo-preserved bovine sperm both in light and fluorescent microscope are presented in Plate 3.3. A s it is observed in the photographs, The acrosomal integrity is severely damaged in the case of calcium ionophore compared to the case of progesterone or db - cAMP. It is probably because of different mechanism of calcium ionophore to affect A R compared to that o f progesterone. 7 1 PLATE 3.3. Calcium ionophore-induced AR. A) Under the fluorescent microscope. B) The same field under the light microscope. The sperm showed by arrows were acrosome-reacted. In the sperm with solid arrows acrosomal content were completely disappeared while in those with unfilled arrows acrosomal content were not disappeared although they were acrosome-reacted. 72 F I G U R E 3.9. The percentages of A R increase following Ca 2 +- ionophore treatment in six bulls (n = 72). Bu l l Numbers e . Values were differences between acrosome reaction in control and after each treatment. \ Values were reported as the mean value ± standard errors of the means. .^ Values with different alphabetical superscripts were statistically different (p < 0.05). . Y-axis started at 80 for clarity. 3.3.4. Correlation of in vivo fertility and A R inducibility of semen samples A s shown in Figure 3.1., 3.7., 3.8., and 3.9., percentages o f A R increases in response to progesterone, d b - c A M P , forskolin, and calcium ionophore treatment o f sperm vary significantly among bulls. When the correlation between in vivo fertility of bulls (90-day non-return rates o f each individual bull) and the ability of their sperm to be acrosome-reacted in effect o f progesterone, d b - c A M P , forskolin and calcium ionophore were assessed, they were not correlated. 73 FIGURE 3 . 1 0 . The percentages o f A R increase following Progesterone, db - cAMP, Ca 2 +-Ionophore, forskolin, and 17 p-estradiol treatment in six bulls (n = 72). coo O 00 1 20 1 00 80 J3 60 O <. 40 20 1 #476 #436 #391 #389 Bu l l Numbers ES Progesterone H D b - c A M P • Ca-Ionophore B Forskolin • Estradiol #444 #461 . Values were differences between acrosome reaction in control and after each treatment. . Values were reported as the mean value ± standard errors o f the means. Based on the results of this study using different A R inducers (Figure 3.10.), the bulls were divided to two groups with respect to their A R inducibilties. Bul ls #389, #391, and #461 had more A R inducibility and Bul ls #444, #436, and #476 had a lower A R inducibility. The average percentages of non-return rates for these two groups were 70% and 69.3%, respectively which were not different. 3.4. DISCUSSION Based on the results of the present study, on an average, sperm motility underwent a sharp drop over the first two hours after thawing, although sperm viability remained largely unaffected. Further incubation did not result in a significant decrease in sperm motility. 74 Several studies have compared the basic traits of semen quality with fertility estimates and correlations have ranged from zero to very high (Saacke, 1982). Budworth et al. (1988) reported a correlation between sperm motility and fertility. In contrast, Bai ley et al. (1994) reported the absence of any correlation between motility parameters and in-vivo fertility o f cryopreserved bovine spermatozoa. In the present study, differences were observed among individual bulls in the percentages of post-thaw motility decrease over the first two hours o f incubation. However, no correlation was found between either post-thaw motility or post-thaw motility drop (over 2-hour incubation in capacitation medium) of cryopreserved semen and in vivo fertility o f the bulls. Viabi l i ty of sperm didn't show any significant decrease over the six hours o f capacitation incubation after thawing. In this study, no significant spontaneous A R was observed during six hours of incubation for capacitation. This was in contrast to the results o f the previous studies in human sperm in which small population o f sperm underwent spontaneous A R over 2-18 hours of incubation in capacitation medium (Parinaud et al, 1992; Lee et al., 1998). The absence of spontaneous A R over time would probably indicate that the cryopreserved sperm is capacitated immediately after the thawing and swim-up preparation. Therefore, the subpopulation of capacitated sperm did not increase over the 6-hour incubation period and neither did the spontaneously acrosome-reacted sperm. When three different concentrations of progesterone (0.1, 1, 10 ug/ml) were used to induce A R in the frozen-thawed bovine sperm, the extent o f A R induced were dose-dependent. This was in complete agreement with previous studies in human sperm (Lee et al., 1998; Blackmore et al., 1990). The concentration of progesterone to induce A R in bovine sperm was found to be 1-10 p.g/ml which is comparable to its physiological concentration in the exact location o f fertilization, ampula. In human, progesterone has also been reported to induce A R 75 at 1-10 ng/ml (Osman et al, 1989; Blackmore et al, 1990; Tesarik, 1992; Sueldo et al, 1993; Lee et al, 1998). Progesterone at 0.1 u.g/ml did not show any significant increase in the percentages of acrosome-reacted bovine sperm as compared with control. Similar results have been also reported in case of human sperm (Tesarik, 1992; Sueldo et al, 1993; Lee et al, 1998). Progesterone at 10 p.g/ml was found to exert its optimum effects in bovine sperm. This concentration has also been previously reported in human sperm as an optimum concentration (Shivaji and Jagannadham, 1992; Lee et al, 1998). In vivo, progesterone can be secreted in relatively high concentrations in cumulus cells and it is also present in tubal f luid to induce sperm A R . The level o f progesterone in the serum of women after ovulation generally ranges from 6-14 ng/ml (0.006-0.01 u.g/mi) (O'Mal ley and Strott, 1991) and it may be too low to affect sperm A R . Similarly the progesterone in human tubal f luid w i l l most l ikely be absorbed or diluted following ovulation and this would l ikely depend on the time after ovulation. Therefore, human tubal f luid may not be that much effective for capacitation or A R after dilution at the site of fertilization (Uhler et al, 1992). Thus, it seems that progesterone produced by the cumulus cells may be the most l ikely source for the sperm A R induction (Blackmore et al, 1990; Uhler et al, 1992). Cumulus cells surround the human oocyte at the very moment o f fertilization. Since progesterone secretion by one single human oocyte-cumulus complex has been measured at roughly 5-15 ng/hour (Hillensjo et al, 1985), the concentration of progesterone within the cumulus matrix could, easily exceed the 1000 ng/ml (1 ug/ml)(Osman et al, 1989; Uhler et al, 1992). This concentration of progesterone has been used in the present study and shown to be effective in the in vitro studies (Osman et al, 1989; Blackmore et al, 1990; Tesarik, 1992; Sueldo et al, 1993; Lee et al, 1998). Indeed, it has been demonstrated that single human cumulus cell matrices (Tesarik, 1985) and fragments (Siiteri et al, 1988) could induce A R in vitro. In 76 conclusion, in the microenvironment surrounding the ovulated egg, the cumulus mass may provide sufficient concentrations of progesterone to induce a multitude of changes in human spermatozoa, resulting in A R (Blackmore et al, 1995; Turner and Me ize l , 1995). Furthermore, it is generally agreed that A R and the initiation of sperm-oocyte fusion can only happen in cells that have been primed by capacitation (Ohzu and Yanagimachi, 1982; llanos and Me ize l , 1983; Parrish et al, 1988; Uhler et al, 1992; Wistrom and Me ize l , 1993; Shi Q-x and Roldan, 1995; Motamed Khorasani et al, in press). The capacitation time for fresh bovine sperm using heparin has been reported to be at least 4 hours (Parrish et al, 1988). Interestingly, no capacitation time was required in the present study for frozen-thawed bovine sperm to be induced by progesterone for A R . This could not be because of the preventive effects o f medium on heparin, because no glucose was used in the T A L P medium to block the heparin effects. Thus, the most l ikely conclusion to be drawn from the present results would be that cryopreserved bovine sperm are capacitated immediately after thawing. This would probably suggest that the subpopulation of sperm capable o f undergoing the capacitation, have underwent this process either in effect o f adding freezing media prior to cryopreservation or in effect o f the changes happening during freeze-thaw procedures. This suggestion would be further supported by the fact that cryopreservation can damage subcellular compartments o f spermatozoa (Creenhalgh, 1990). Furthermore perturbations in membrane structure during cryopreservation (Papadopoulos, 1993) may alter the ability of spermatozoa to maintain ion concentration gradients (Caffrey et al, 1979; Sernia et al, 1982). It may also affect the N a + / K + ATPase activity (Bramley and Ryan, 1978) and finally can lead to potentially lethal increases in intracellular C a 2 + concentration (Caffrey et al, 1979). The increase in intracellular concentration is the end result o f capacitation. Thus A R could be induced in frozen-thawed sperm in the presence of A R inducing factors, as signal transduction pathways, downstream to intracellular C a increase, would be turned on. 77 Since capacitation is a prerequisite for A R , a relationship must exist between capacitation rate and fertility. Unfortunately, there is no direct method available to differentiate between capacitated and non-capacitated spermatozoa in vitro. Presently, assessment o f capacitation is based on subjective and indirect methods such as hyperactivated motility, induction o f A R 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). In the present study, the possibility o f uti l izing the induced A R as a laboratory test to predict the in vivo fertility o f breeding bulls was also considered. The results of this study indicated no relationship between the percentages of in vivo fertility o f the bulls and the percentages of induced A R of cryo-preserved sperm. In contrast, the existence of a strong linear relationship between the ability of fresh bull spermatozoa to undergo heparin-induced A R in vitro, and non-return rates, has been reported by others (Ax et al., 1985; Whitf ield and Parkinson, 1992). In this study, it was found that the inducing effect of 17 P-estradiol was too small to be Practical although significantly differed from ethanol as control. In human 17 P-estradiol has been reported to have very little effect on A R induction at 2-10 ug/ml (Margalioth et al, 1988; Thomas and Meize l , 1989; Meize l et al, 1990; Blackmore et al, 1990; Shivaji and Jagannadham, 1992). It has been wel l established that only progesterone and 17 a -hydroxyprogesterone could induce the A R and the influx of C a 2 + into sperm, But this is not the case for other steroids (Margalioth et al, 1988; Thomas and Meize l , 1989; Blackmore et al, 1990; Me ize l et al, 1990; Shivaji and Jagannadham, 1992). The exact reason as to why only certain steroids interact with spermatozoa and induce physiological effects such as the A R are not known (Shivaji and Jagannadham, 1992). The binding of progesterone to human sperm has 78 been known for a long time (Cheng et al, 1981), and is probably mediated by a receptor in the plasma membrane (Blackmore et al., 1990; Motamed Khorasani et al, in press). In the present study, one u,g/ml of exogenous cholesterol was found to block progesterone-induced A R almost completely. Similar results were reported in human sperm (Cross, 1996; Lee et al, 1998). Cholesterol was also found to inhibit the progesterone- and db-cAMP- induced A R in bovine sperm to the same degree as in human sperm. P-sitosterol, a plant analog of cholesterol with no known receptors in the mammalian cells (Vander kraak and Mac Latchy, 1995; Fernandes et al, 1996; Nagy et al, 1996; Mac Latchy, 1997; Nguyen et al, 1999; Zakaria et al, 1999) (Appendix E) , inhibited A R in an identical manner. Thus, it is unlikely that cholesterol exerts its action via a specific receptor-mediated pathway. Instead, cholesterol and p-sitosterol exert a more general effect on the sperm plasma membrane. A s a result, sperm surface receptors would be masked from interacting with the corresponding ligands to activate the signal transduction pathway leading to A R as in the case of progesterone and its receptors. Similar results have been reported in human by Motamed Khorasani et al. (in press). So the mechanisms of cholesterol action seem to be similar both in human and in bovine sperm. A s indicated previously, it was found that progesterone- and db-cAMP-induced A R were significantly inhibited by cholesterol. The cellular events that culminate in A R have been shown to involve at least two second messenger pathways (Zaneveld et al, 1991) one of which involves the generation of the second messenger, c A M P (by the amplifying enzyme adenylate cyclase), which ultimately activates cAMP-dependent kinase ( P K A ) (De Jonge et al, 1991). In this study, A R induction by the c A M P analogue, db - cAMP, was significantly inhibited by exogenous cholesterol (Figure 3.6.). This would indicate that db - cAMP was prevented by cholesterol from transporting through cell membrane to activate the P K A signal transduction 79 pathway. The same scenario may happen in the case of progesterone. These observations would imply that cholesterol exerted its effect upstream of adenylate cyclase by blocking ligand-receptor interaction on the sperm surface. The presence of progesterone receptors in the human sperm plasma membrane has been reported by several previous studies (Blackmore et al, 1990, 1991; Meize l and Turner, 1991; Sabeur et al, 1996; Ai tken et al, 1996; Alexander et al, 1996; Meize l , 1997; Cheng et al, 1998). It has been demonstrated that the binding of progesterone to its surface receptors is followed by receptor aggregation (Tesarik and Mendoza, 1993a; Motamed Khorasani et al, in press), stimulation of rapid C a 2 + influx and A R (Tesarik et al, 1992; Wistrom and Meize l , 1993; Ai tken et al, 1996; Motamed Khorasani et al, in press). The finding of d b - c A M P effects on A R being inhibited by cholesterol in this study, therefore, further support the hypothesis that cholesterol exert its effects at the level of sperm plasma membrane v ia altering its fluidity, and consequently preventing progesterone receptors to be exposed. It was reported previously (Tesarik et al, 1996), that the addition o f progesterone produced a rapid transient [Ca 2 + ] increase in a minor population o f spermatozoa. In one third of this population, this initial [Ca 2 + ] increase was followed by a secondary [Ca 2 + ] increase 2-10 minutes after addition o f progesterone and finally acrosomal exocytosis. The initial protein tyrosine kinase-independent C a 2 + response could be induced by progesterone in both non-capacitated and capacitated spermatozoa, whereas the generation o f the secondary protein tyrosine kinase-dependent response could only be generated during in vitro capacitation (Tesarik et al, 1996). Further research is needed to study why only a portion of the sperm have their progesterone receptors exposed and its relevance in the fertilization capacity of the human sperm. 80 In conclusion, the results o f this study suggest that cholesterol inhibits A R by modifying the surface structure of the sperm plasma membrane, thereby, masking the surface receptors for progesterone binding. The results of this study also suggest that reversible cholesterol removal from the sperm plasma membrane and the subsequent progesterone receptor exposure are inter-related important events during the process of sperm capacitation. It remains to be explained why only a minor population of sperm are selected to have the initial progesterone-induced response. In case of forskolin-induced A R , cholesterol only partially inhibited the forskolin effect on A R (21.2% inhibition). Forskolin with molecular weight o f 410.5 can probably bypass the cell membrane (without any receptor in the cell surface) because of its hydrophobic specifications and the addition of cholesterol to the medium can possibly decrease the loss of cholesterol from sperm plasma membrane and increase the rigidity o f membranes. But why forskolin effects can not be inhibited by cholesterol is not clear and more investigation is needed to be done. In contrast to its inhibitory effects on progesterone and d b - c A M P , cholesterol could not exert any inhibition in the presence of calcium ionophore. Because of essential role of ion gradients in active transport and energy conservation, compounds that collapse the ion gradients across cellular membranes are detrimental to the cell and prevent any reversible capacitation. Compounds that shuttle ions across membranes in this way are called ionophores, literally "ion-bears" (Lehninger et al., 1993). It has been found that a hallmark o f the A R process is its absolute requirement of extracellular C a 2 + (Yanagimachi and Usu i , 1974; Fraser, 1987; Okamura et al, 1991; Visconti and Kopf, 1998). Hence C a 2 + is regarded as the key regulator of A R . On the other hand, high levels of calcium ion is toxic for the cells (Caffrey et al., 1979) and may also adversely affect spermatozoa survival (Luck, 1988). Ionophores also uncouple oxidative phosphorylation. These agents bind to inorganic ions and surround them 81 with hydrophobic moieties. The ionophore-metal ion complexes can pass easily through membranes and uncouple A T P synthesis and oxidative phosphorylation in the mitochondria. A s a consequence of A T P drop, the sperm motility would drop. Based on these reports, the effects o f ionophores are not mediated via a surface receptor and therefore its action on A R is not influenced by the presence of cholesterol, as they disrupt the l ipid structure o f biological membranes (Klausner et al., 1979). Moreover C a 2 + ionophore has been reported to obviate or by-pass the need for capacitation by forming a lipophilic complex with calcium ions, which results in the rapid transport o f the C a 2 + ions across the sperm plasma membranes. The end result of this C a 2 + influx is the rapid precipitation of an A R and the concomitant generation of a fusogenic equatorial segment (Talbot et al., 1976; Shams-Borhan and Harrison, 1981; Smith et al., 1983; Dvorak et al, 1984). Therefore, A R induced by calcium ionophore is not supposed to be affected by the presence of cholesterol, as it has been shown in this thesis. 3.5. CONCLUSIONS Results of this investigation clearly demonstrate that the frozen-thawed bovine sperm are capacitated immediately after thawing and A R can be induced without further incubation for capacitation. Further more the observed effective ability of progesterone and d b - c A M P to induce A R in frozen bovine sperm, would further strengthen the future possibility of progesterone and db -cAMP being utilized to improve the IVF success rates. N o relationship was found between post-thaw sperm motility, post-thaw motility decrease over time, induced A R and in vivo fertility of bulls. It is concluded that post-thaw sperm motility or the percentage of induced A R may not be reliable markers for predicting field fertility of breeding bulls. Cholesterol was found to effectively inhibit the effects of progesterone and d b - c A M P on A R in the same manner that P-sitosterol did. It may be concluded that the mechanisms of 82 A R inhibition by cholesterol and P-sitosterol, a general plant sterol, in bovine and human sperm are identical. Although the exact cellular mechanism of cholesterol on sperm remains to be elucidated, the alteration of membrane fluidity and consequently, the fusibility of cell membranes may be the key factor. Further studies would be required to demonstrate i f cholesterol inhibition o f A R could be used in fertility regulation in vivo. Similarly, it remains to be shown i f progesterone-induced A R could be used in the improvement o f in vitro fertilization rates in bovine or human IVF . 83 CHAPTER 4 E F F E C T S O F P R O G E S T E R O N E AND C H O L E S T E R O L O N S P E R M A N D O O C Y T E I N T E R A C T I O N IN BOVINE IN VITRO F E R T I L I Z A T I O N S Y S Y T E M A s it has been demonstrated in Chapter three, acrosome reaction (AR) o f bovine sperm can be induced by progesterone (P 4 ) , whereas cholesterol (CH) inhibits P 4- induced A R . The objectives of this study were to determine the effects of exogenous P4 and C H on zona-binding o f bovine sperm and bovine in vitro fertilization (IVF). For the zona-binding assay, frozen-thawed sperm were washed in modified Tyrodes 1 and final concentration was adjusted to 0.2-0.5 x 10 6 sperm/ml. Sperm were pre-incubated with P 4 (10 p.g/ml, 30 min), either in the presence or absence o f C H (1 ng/ml) at 38.5°C, 5% CO2. Sperm were co-incubated with cumulus-free oocytes for 18 h (30 oocytes/treatment). Fol lowing washing, f ixing, and staining steps, the number o f sperm attached to oocytes were counted under a fluorescent microscope. For the I V F studies, 30 minutes prior to the addition o f matured oocytes, sperm (0.2 x 10 6 sperm/ml) were separately pre-treated with P 4 (10 ng/ml), C H (1 ug/ml), combination of P4 and C H , and cyclodextrin (10 ug/ml) (120 oocytes/treatment). Sperm exposed oocytes were then washed twice and incubated in culture medium (modified Tyrodes' supplemented with 10% serum) at 38.5°C and 5% CO2. Cleavage rates, as an indicator of fertilization, were recorded after 72 hr. Progesterone increased the number of spermatozoa bound to oocyte by 73.9 ± 3 . 7 compared to control in which the number o f bound sperm was 45.8 + 2.8. When the sperm were treated with C H alone or the combination of P4 and C H the number of bound sperm was 45.3 + 1.8 and 46.2 + 3.3, respectively. Progesterone also increased the percentages o f cleavage rate by 42.5 ± 3.8% compared to 22.5 ± 2.1% in control, 18.3 ± 2.8% for C H , 84 20.8 ± 2.0% for P-cyclodextrin, and 17.5 ± 1.7% for P 4 and C H combination. It is concluded that P 4 treatment o f sperm immediately prior to fertilization may improve mammalian I V F success rate. The presence of C H in addition to P 4 reversed the cleavage rate and zona-bound sperm enhancement caused by P 4 . 4 . 1 . I N T R O D U C T I O N Although the precise time at which the A R occurs during the fertilization process is controversial, it is generally agreed that it has to take place before the sperm can penetrate the zona pellucida (ZP) and fuse with the oocyte plasma membrane(Yanagimachi, 1988). In deed, according to the current dogma, capacitated and acrosome-intact spermatozoa initiate binding to the zona pellucida (Yanagimachi, 1994). Although the zona pellucida is considered the prime physiological inducer o f A R , previous studies have shown a low incidence of A R in zona pellucida-bound stallion spermatozoa after 1-hour in vitro binding (Ellington et al., 1993; Meyers et al., 1996; Cheng et al., 1996). This low incidence suggests that, besides the zona pellucida glycoproteins, another major factor might be responsible for A R . It has been speculated by many researchers that this major factor which should be present at the site and at the very moment o f fertilization, is probably progesterone (Laufer et al, 1984; Zorne et al, 1982; Margalioth et al, 1988; Sueldo et al, 1993). Moreover, major inducing effects o f follicular f luid have been reported to be due to its progesterone and 17-hydroxyprogesterone levels (Osman et al, 1989). After ejaculation, the spermatozoa move from the seminal plasma fluid, an environment low in progesterone (0.3-0.7 ng/ml) (Abdalla et al, 1981) through the uterus to the fallopian tube where progesterone concentration after ovulation are in the range of 30-300 ng/ml (Zom et al, 1982). The oocyte and the surrounding cumulus cells produce 600-800 ng of progesterone (Laufer et al, 1984) during the 85 first day after ovulation. Therefore, when the spermatozoa reach the oocyte in the tube, the surrounding fluid is rich with progesterone (Margalioth et al, 1988). A s it has been discussed in the previous chapter of this thesis, progesterone has been shown to induce the A R in human (Foresta et al, 1993; Meize l and Turner, 1991; Lee et al, 1998), mouse (Roldan et al, 1994), pig (Melendrez et al, 1994), and bovine spermatozoa, as it has been shown in the current study. Furthermore, an early event involved in the A R is an obligatory increase in free cytosolic calcium (Stock and Fraser, 1989; Blackmore et al, 1990; Bald i et al, 1991). It has been reported that preovulatory human follicular f luid enhances the influx o f calcium through the sperm cell membrane (Thomas and Meize l , 1988), the A R percentages (Tesarik, 1985; Suarez et al, 1986; Siiteri et al, 1988; Siegel et al, 1990). Again progesterone in the follicular fluid has been speculated and actually shown to be responsible for this calcium influx (Blackmore et al, 1990; Baldi et al, 1991; Foresta et al, 1993; Meize l and Turner, 1991; Emi l iozz i etal, 1996). Considering all these facts, it was hypothesized that pretreatment o f spermatozoa with exogenous progesterone may result in an increase in the number of capacitated and acrosome-reacted sperm in the vicinity o f the oocyte and consequently an increases in the number of sperm bound to oocyte and fertilized oocytes. Based on the previous results of this study the optimum inducing effects o f progesterone can be reached in 30 minutes, sperm pre-treated progesterone for 30 minutes should immediately be co-incubated with oocytes. It was hypothesized that under the mentioned conditions, because of the progesterone-induced capacitation, hyperactivity, and concomitant progesterone- and zona-induced A R , higher rates of cleavage would be achieved. This experiment was carried out towards two objectives. First elucidating the effects of progesterone and cholesterol pre-treatment of spermatozoa in bovine IVF system and second, the role o f these two chemicals on sperm-zona interactions. For the first part four trials were 86 carried out: 1) The standard IVF procedure with B O medium and five mi l l ion sperm/ml concentration, 2) The standard I V F procedure with B O medium but with a range of sperm concentration from 0.1 to 5 mi l l ion sperm/ml, 3) The standard I V F procedure with B O medium but with the best practical sperm concentration for this study (0.2 mi l l ion sperm/ml), 4) The I V F procedure by Sivakumaran et al, 1993 with T A L P medium and 0.2 mi l l ion sperm/ml. 4.2. M A T E R I A L S A N D M E T H O D S 4.2.1. Chemicals and culture media The following chemicals were purchased from Sigma chemical company, St. Louis, M O , U S A : bovine serum albumin (0.3% B S A ) , progesterone p-cyclodextrin complex (water soluble progesterone), cholesterol p-cyclodextrin complex (water soluble cholesterol), P-cyclodextrin, heparin sodium salt (heparin sulfate, 1000 IU/ml), sodium bicarbonate, sodium chloride, potassium chloride, sodium phosphate monobasic, sodium lactate, calcium chloride, magnesium chloride, H E P E S , sodium pyruvate, gentamicin, penici l l in (100,000 IU), streptomycin, Dulbecco's phosphate buffered saline (PBS), foll icle stimulating hormone (FSH), caffeine sodium benzoate, insulin, Hoechst 33342 nuclear stain, and 2.5% glutaraldehyde. T C M 199 was manufactured by Gibco B R L , L i fe Technologies. 4.2.2. Semen samples Semen samples from six Holstein bulls were used in this study (#444, #389, #461, #391, #476, #436). Based on the previous study (Chapter 3), the bulls were divided into two groups (three bulls in each group), one with higher A R inducibility (#389, #391, and #461) and the other with lower A R inducibility (#444, #436, and #476). Semen samples of three bulls in each 87 group were pooled and this was done for three different ejaculates of bulls yielding a total o f six experimental groups for this study. 4.2.3. Standard IVF procedures using B O medium The standard I V F procedures carried out using B O medium and normal sperm concentration (5 x 10 6 sperm/ml) and the procedures are presented in this section. A n illustrative presentation o f the protocol is presented in Appendix D. 4.2.3.1. Collection of Ovaries Ovaries were collected from a local slaughterhouse (ovaries o f culled caws with unknown pedigree) and placed in an insulated container to maintain the temperature at 30°C. They were brought to the laboratory within 2 hours. Upon arrival ovaries were washed in 30-32°C (Sato et al, 1977; Shioya et al, 1988; schleger et al, 1990) collection media (Appendix A ) . Ovaries were placed in a beaker containing collection media o f 32°C. The beaker was then placed in a water bath to maintain the temperature at 32°C. 4.2.3.2. Aspiration of Oocytes Oocytes were collected from follicles with the diameter o f 1-6 mm ( Mot l ik and Fulka, 1986; Tan and L u , 1990; Lonergan, 1990) by aspiration with an 18 gauge (1.5 inch) needle and a 5 ml syringe (Sreenan, 1968; Hunter et al, 1972; Leibfried and First, 1979; X u et al, 1987, 1988). Aspirated cumulus-oocyte complexes were washed twice in oocyte aspiration media to remove debris (Appendix A ) . 88 4.2.3.3. Oocyte Evaluation Cumulus-oocyte complexes were transferred to a graded petri dish for microscopic evaluation. Oocytes with homogeneous, evenly granulated cytoplasm and a compact intact cumulus mass (Plate 4.1.) were selected (Leibfried and First, 1979; Greve and Madison, 1991; Brackett and Zuelke, 1993). Oocyte pick up was done by mouth pipeting with a micrometer diameter sterilized Pasteur pipette, under a dissecting microscope. 4.2.3.4. In Vitro Maturation Selected cumulus-oocyte complexes were transferred to mini-petri dishes containing maturation media at 38.5°C (Appendix A ) (Lu and Gordon, 1987; L u and Polge, 1992). The mini-petri dishes containing selected oocytes were incubated at optimal conditions o f 38.5°C (Lenz et al, 1983b) and 5% CO2 in air (Azambuja et al, 1993) for a 16-18 hour period (Lu et al, 1987a, 1987b, 1988a, 1988b, 1988c; Monaghanef al, 1993). 4.2.3.5. Sperm preparation Cryopreserved semen samples were provided Westgen, Mi lner, Bri t ish Columbia, Canada. Frozen straws were thawed in a 37°C water bath for 40 seconds. Quantitative motility was determined by counting both motile and immotile spermatozoa in at least 10 separate and randomly selected fields. A t least 100 spermatozoa were counted. The percentage o f motile spermatozoa was calculated from the mean percentage motility for all fields counted (World Health Organization, 1992). Thawed semen samples were then centrifuged twice (Lue et al, 1987b; Ijaz and Hunter, 1989; L u , 1990) with Caf f -BO medium at 500 x g for 5 minutes. The sediment was resuspended in Caf f -BO plus B S A and heparin (Ca f f -BO-BSA, Appendix A ) (Parrish et al, 1984; Mi l le r and A x , 1988, 1989a, 1989b, 1990). The concentration o f sperm 89 was determined using hemocytometer, after immobil izing the sperm with 3% N a C l solution. Final sperm concentration was adjusted to 5 x 10 6 sperm/ml (Brackett et al, 1982; Bousquet et al, 1984; Lambert et al, 1986; Parrish et al, 1986). The incubation was carried out at 38.5°C and 5% CO2 in humidified air (100% relative humidity). 4.2.3.6. In Vitro Fert i l izat ion Sperm manipulation was carried out with frozen-thawed semen according to the techniques used in Animal Reproductive Biotechnology Laboratory (Yamaguchi University, Japan). Over night matured oocytes were washed twice in C a f f - B O - B S A media (Appendix A ) . Three 20-u.l aliquots of sperm suspension were placed in a sterilized micro-petri dish followed by a covering of mineral oi l and final injection o f three 80-ul aliquots of sperm suspension to each droplet. Matured oocytes were then selected and added to each droplet (20 oocytes/100 ul-droplet o f sperm solution). The oocyte-sperm suspension was incubated at 38.5°C (Lenz et al, 1983a; Cheng et al, 1986; Hunter, 1992) and 5% C 0 2 in air for a 16-18 hour period (Lu et al, 1987b). 4.2.3.7. In Vitro Cu l tu re Fol lowing incubation of sperm and oocytes for 16-18 hours, oocytes were washed twice in culture media (Appendix A ) to remove excess sperm. Oocytes were then transferred into 4-wel l dishes containing 500 ul of culture media/well. Culture media containing oocytes was covered with mineral oi l and incubated for 24 hours at 38.5°C and 5% CO2 in air. Due to the nutrient requirements o f developing embryos, culture media was changed every 72 hours, according to the techniques used in Animal Reproductive Biotechnology Laboratory (Yamaguchi University, Japan). 90 Forty-eight hours after co-culture, oocytes were examined for cleavage to assess fertilization rate. At days 7, 8, and 9 of the experiment, the blastocyst formation rate was also assessed. The percentages of cleaved embryos and blastocyst formation were estimated and were compared among different treatments. The photographs of 2-cell, 4-cell, 8-cell embryos, as well as hatching and blastocysts are presented in Plates 4.2, 4.3, 4.4, 4.5, and 4.6, respectively. 91 92 PLATE 4.5. H a t c h i n g b o v i n e e m b r y o ( Z o n a p e l l u c i d a s h o w n by an a r row ) . 93 PLATE 4.6. Hatched bovine embryo. 4.2.4. Effects of progesterone and cholesterol on cleavage and blastocyst format ion rates on standard in vitro fert i l izat ion Oocytes were recovered and sperm preparation was carried out as described previously. A total of 450 oocytes were used and for each of replicates 90 oocytes were studied (30 oocytes/treatment, three replications). Sperm concentration was adjusted to 5 x 10 6 sperm/ml. Five different treatments were carried out. Sperm were pretreated with progesterone (10 fig/ml), cholesterol (1 ug/ml), and combination of progesterone and cholesterol, 30 minutes before co-incubation with oocytes (cholesterol was added first and 10-15 minutes later progesterone was added for an additional 30 minutes). The diluent for progesterone and cholesterol, (3-cyclodextrin, was added to sperm at a comparable concentration (10 tig/ml) for 30 minutes before co-incubation with oocytes (as a negative control for progesterone and 9 4 cholesterol). A s a general control for treatments, sperm and oocytes were co-incubated without adding any of the above. After sperm treatment, sperm and oocytes were co-incubated and the rest of the procedures were carried out as described in section 5.3.3. The cleavage rate was estimated as an indicator for fertilization and so was the blastocyst formation rate. 4.2.5. Effects of sperm concentration on cleavage and blastocyst formation rates in vitro Based on the results of the previous study, it was decided to find the best sperm concentration to demonstrate the possible effects of progesterone and cholesterol. A total o f 480 oocytes were used. For each o f replicates 160 oocytes were studied (60 oocytes/treatment, 3 replications). Oocyte were prepared and frozen sperm were thawed and as described previously. Final sperm concentration was set to 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2.5, and 5 x 10 6 sperm/ml. Sperm and oocytes were then co-incubated and the rest o f the procedures were carried out as it is explained in section 5.3.3. The cleavage rate was estimated as an indicator for fertilization and so was the blastocyst formation rate. 4.2.6. Effects of progesterone and cholesterol on cleavage rate in vitro (with 0.2 x 106 sperm/ml, in B O medium) Oocyte were prepared and frozen sperm were thawed and as explained previously. In this study, a total of 600 oocytes were used. For each replicate 100 oocytes were studied (120 oocytes/treatment, replicated 6 times). Based on the results of A R studies (section 3.4.4.), the bulls were divided to two groups with respect to their A R inducibilties. Bul ls #389, #391, and #461 had more A R inducibility and Bul ls #444, #436, and #476 had a lower A R inducibility. In I V F studies also, the bulls 95 were divided into the same two groups and effects of progesterone and cholesterol were studied. In each group the semen of the three individuals were pooled and treatments were carried out in three replicate experiments (Total of 6 replicate experiments). Based on the previous results, sperm concentration was adjusted to 0.2 x 10 6 sperm/ml (20,000 sperm/drop or 1000 sperm/oocyte). F ive different treatments were carried out for sperm before co-incubation with oocytes, control, progesterone (10 u,g/ml), cholesterol (1 u,g/ml), p-cyclodextrin (10 ng/ml), and a combination of progesterone and cholesterol, as it is mentioned in section 5.3.4. After sperm treatment, sperm and oocytes were co-incubated and the rest o f the procedures were carried out as it is explained in section 5.3.3. The percentages of cleaved embryos were estimated as an indicator for fertilization, and were compared among different groups o f treatment. The results were compared in both groups o f pooled semen, those with lower and those with higher A R inducibility. 4.2.7. Effects of progesterone and cholesterol on cleavage rate in vitro (with 0.2 x 106 sperm/ml, T A L P medium) A s the expected results were not accomplished in the previous I V F experiments, even with the best sperm concentration for this study, another set o f experiments with another media was carried out. This time, the procedure reported by Sivakumaran et al, 1993 was used. A n illustrated summary o f this protocol is presented in Appendix C . A total of 600 oocytes and For each replicate 100 oocytes were studied (120 oocytes/treatment, 6 replications). Aspiration and selection of cumulus-oocyte complexes were carried out as mentioned in section 4.3.3.2 and 4.3.3.3. Selected cumulus-oocyte complexes were transferred to mini-petri dishes containing Ham's F-10 (Gibco, Grand Island, N Y , U S A ) supplemented with 10% estrus 96 cow serum. The mini-petri dishes were incubated at optimal conditions o f 38.5°C and 5% CO2 in humidified air and 100% R H for a 26 hour period. Over-night matured oocytes with good quality were collected and used for I V F in Fer t -TALP medium (Appendix B) . Based on the results of A R studies (section 3.4.4.), the bulls were divided to two groups with respect to their A R inducibilties. Bul ls #389, #391, and #461 had more A R inducibility and Bul ls #444, #436, and #476 had a lower A R inducibility. In I V F studies also, the bulls were divided into the same two groups and effects o f progesterone and cholesterol were studied. In each group the semen of the three individuals were pooled and treatments were carried out in three replicate experiments (Total o f 6 replicate experiments). Frozen straws were thawed in a 37°C water bath for 40 seconds. Mot i l i ty was determined by counting spermatozoa in at least 10 separate and randomly selected fields until at least 100 spermatozoa were counted (World Health Organization, 1992). Thawed semen samples were then washed twice in modified Tyrode's medium ( S P - T A L P medium, P H 7.4, Appendix B) at 500 x g for 5 minutes (Parrish et al., 1988). The sediment was resuspended in Tyrode's medium supplemented with 10 p.g/ml heparin sulfate (Fer t -TALP; Appendix B) . Concentration o f sperm was estimated using hemocytometer, after immobil izing the sperm with 3% N a C l solution. Final sperm concentration was set to 0.2 x 10 6 sperm/ml (based on previous results). F ive different treatments were carried out in this experiment as it is mentioned in section 5.3.4. The incubation condition remained the same as it was mentioned for previous experiments (38.5°C and 5% C 0 2 in humidified air, 100% RH) . Over night matured oocytes were washed twice in Fer t -TALP. Three 20 u l aliquots of sperm suspension were placed in a sterilized micro-petri dish followed by a covering of mineral o i l and final injection o f three 80 u l aliquots of sperm suspension to each droplet. Matured and washed oocytes were then selected and added to each droplet. Twenty oocytes were placed in 97 each 100 iil-droplet o f sperm preparation (20,000 sperm/drop or 1,000 sperm/oocyte). The oocyte-sperm suspension was incubated at 38.5°C and 5% CO2 in air for a 15-hour period. After fertilization (15 hours), the fertilized oocytes were washed twice in Ham's F-10 (Gibco, Grand Island, N Y , U S A ) supplemented with 10% fetal bovine serum (FBS) , to remove excess sperm. Ferti l ized oocytes were then transferred into 4-well dishes containing 500 u l o f Ham's F-10 supplemented with 10% F B S . Culture media containing oocytes was covered with mineral o i l and incubated at 38.5°C and 5% CO2 in air. Culture media was changed every 96 hours. Forty-eight hours after fertilization, oocytes were examined for evidence of cleavage to assess oocyte fertilization rate. The percentages of cleaved embryos were estimated and were compared among different groups of treatment. The results were compared in both groups of pooled semen, those with lower and those with higher A R inducibility. 4.2.8. Effects of Progesterone and Cholesterol on Sperm-Zona Binding Ability A s mentioned previously, the bulls were divided into two groups (three bulls in each group). One group with higher A R inducibility (#389, #391, and #461) and one group with lower A R inducibility (#444, #436, and #476). In this experiment, only the first group was studied. Three semen samples from three bulls in this group were then pooled and this was done for three different ejaculates o f bulls to get three experimental groups. A total of 240 oocytes were used. For each replicate 40 oocytes were studied (30 oocytes/treatment, 3 replications). Ovaries were collected from a local slaughterhouse (from culled cows). Ovaries were frozen for at least 24 hours in order to remove cumulus cells. Methods for recovering oocytes from frozen ovaries for sperm-oocyte interaction studies have been reported (Wheeler and Seidel, 1987; Ell ington et al, 1993; Mi l le r and A x , 1995). Antral follicles with a diameter o f 4 98 to 8 mm were collected from frozen-thawed ovaries by aspiration with an 18 gauge (1.5 inch) needle and a 5 m l syringe. Aspirated cumulus-oocyte complexes were recovered from the follicular f luid, collected in Dulbecco's phosphate buffered saline (PBS) supplemented with 0.4% B S A , and vortexed for 2-4 minutes to remove cumulus cells. The denuded oocytes were then washed with P B S and kept at room temperature until they were incubated with sperm (Fazeli et al, 1993). Frozen sperm were prepared as mentioned previously. Fol lowing washing, final sperm concentration was set to 0.2 (sperm concentration used in this study) and 5.0 x 10 6 (sperm concentration normally used in bovine IVF) sperm/ml. Four different treatments were carried out in this experiment. Sperm were pretreated with progesterone (10 p.g/ml), cholesterol (1 p.g/ml), and combination of progesterone and cholesterol, 30 minutes before co-incubation with oocytes (cholesterol was added first and 10 minutes later progesterone was added for an additional 30 minutes). A s control for treatments, sperm and oocytes were co-incubated without adding any of those chemicals. The incubation condition maintained at 38.5°C and 5% C 0 2 in humidified air and 100% R H . 4.2.8.1. Z o n a b ind ing assay Sperm-zona binding assay (Appendix G) was performed based on earlier works by Fazeli et al. (1993). Four 20-ul aliquots o f sperm suspension were placed in sterilized micro-petri dishes followed by a covering of mineral oi l and final injection o f four 30-ul aliquots o f sperm suspension to each droplet. Cumulus-free bovine oocytes were used without selection (10 oocyte/droplet). The oocyte-sperm suspension was incubated at 38.5°C, 5% C 0 2 in air, and 100% R H for a 4-5 hour period. A t the end of 4 hours, the oocyte-sperm complexes were picked in a 100 u l eppendorf pipette tip and rinsed 10 times with P B S to remove loosely 99 attached sperm. Wi th 5 rinses, the number of sperm bound to the zona remained high, bringing difficulties in counting. The washed oocytes were then fixed in 2.5% glutaraldehyde in P B S (for 10 minutes), washed in P B S , stained with Hoechst 33342 nuclear stain (1 mg/ml in P B S ; Sigma, St. Louis, M O , U S A ) for 10 minutes, and washed with P B S . They were 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 N ikon U V - 2 A filter combination (excitation filter o f 330-380; barrier filter o f 420 nm). The number o f sperm bound to the zona pellucida o f the oocytes were easily counted under the fluorescent microscope (x 200) as they were brightly fluorescent (plate 4.8.). The effects o f progesterone and cholesterol on number of sperm bound to zona pellucida were assessed for both sperm concentration used in the study. 4.2.9. Statistical analysis Statistical analysis employed analysis of variance ( A N O V A ) for between-group comparison followed by pairwise comparison when the overall F-values corresponded to a p value o f less than 0.05. Bonferroni modification o f the F-test was carried out to consider the type 1 error in the comparison. In all experiments, results were reported as the mean values for each set o f data ± standard error o f the means (S.E.) and the level o f statistical significance was defined at a p value of less than 0.05. 4.3. R E S U L T S 4.3.1. Preliminary studies with progesterone and cholesterol When sperm (5 x 10 6 sperm/ml) were pre-treated with optimum concentrations of progesterone and cholesterol before co-incubation with oocytes, no significant changes in the 100 percentages o f cleavage and blastocyst formation rates (Table 4.1.) were observed. It was considered that the high concentration o f sperm was probably the reason for absence o f the progesterone and cholesterol treatment effects. T A B L E 4.1. Effects o f progesterone and cholesterol on cleavage and blastocyst formation rates using B O medium (sperm concentration o f 5 x 1 0 6 sperm/ml and three replicate experiments). Cleavage Rate (%) Blastocyst Formation (%) Control 83.3 ±5.8 {90f 42.2 ± 9.7 (V Progesterone (10 ng/ml) 82.2 ± 6.8 (90f 42.2 ± 2.9 (V Cholesterol (1 ng/ml) 78.9 ± 6.8 (90f 37.8 ±7.8 («0 b Cyclodextrin (10 ng/ml) 83.3 ±9.6 {90f 35.6 ±7.3 (V Progesterone and Cholesterol (10 ng/ml, 1 ng/ml) 84.5 ±4.0 (90f 51.1 ±2.2 (90? . a total of 450 oocytes were used (in three replications). \ Values were reported as the mean value ± standard errors of the means. .^ Values with different alphabetical superscripts in each column are statistically different (p < 0.05). (). Number of oocytes/treatment in brackets (Replications = 3). 4.3.2. Sperm concentration and its effects on cleavage and blastocyst formation rates In order to determine the effects of progesterone and cholesterol, different concentrations of sperm were studied in the existing IVF system and the least concentration at which cleavage and blastocyst formation rates are acceptable was chosen for the rest o f the study. When different concentrations o f sperm (0.1, 0.2, 0.3, 0.4, 0.5, 1, 2.5, and 5 x 10 6 sperm/ml) were co-incubated with oocytes, a sudden drop in cleavage rate and blastocyst formation rates was observed at the sperm concentration below 0.2 x 10 6 sperm/ml (p < 0.05 compared to the concentration of 0.1 x 10 6 sperm/ml) (Figure 4.1). 101 F I G U R E 4.1. Effects of sperm concentration on the percentages o f cleavage rate and blastocyst-formation in bovine IVF system (in three replicate experiments) • % Cleavage • % Blastocyst 0.1 0.2 0.3 0.4 0.5 1 2.5 5 Sperm Concentration (Mil l ion/ml) . A total of 480 oocytes were used. (160 oocytes/replicate, 60 oocytes/treatment, and three replicates). * . Values were reported as the mean value ± standard errors of the means. \ Value was significantly different from that of 0.1 x 106 sperm/ml (p < 0.05). .^ Value was significantly different from that of 0.1 x 106 sperm/ml (p < 0.0000001). This drop was more obvious in the blastocyst formation rates, as at 0.1 x 10 6 sperm/ml concentration, the cleavage rate was 8.87 ± 1.1% while the blastocyst formation rate was only 1.10 ± 1.1%. In contrast, at 0.2 x 10 6 sperm/ml concentration, the cleavage rate was 24.43 + 4.4% while the blastocyst formation rate was only 4.40 + 1.1%. Indeed with at 0.2 x 10 6 sperm/ml concentration, the cleavage rate and blastocyst formation rates increased three times and four times as much as their percentages in 0.1 x 10 6 sperm/ml concentration o f sperm. 102 Hence, 1000 sperm/oocyte (0.2 x 10 6 sperm/ml) concentration was considered as the best for the rest o f this study in order to magnify the partial effects of progesterone and cholesterol in I V F system (Figure 4.1.). 4.3.3. Progesterone and cholesterol effects on cleavage rate (in B O medium) When I V F results were compared for the two groups of bulls (#389, #391, and #461 with higher A R inducibility and Bul ls #444, #436, and #476 with a lower A R inducibility), progesterone treatment did not increase the cleavage rates in those with higher A R inducibility. Cholesterol did not have any effect on cleavage rates in this group (Figure 4.2.). The cleavage rates in control, progesterone, cholesterol, P-cyclodextrin, and combination of progesterone and cholesterol were 41.7 ± 4.4%, 50.0 ± 5.8%, 43.3 ± 4.4%, 45.0 + 5.0%, 43.3 ± 6.0%, respectively (Figure 4.2.). When IVF results were compared for the group o f bulls with a lower A R inducibility (Bulls #444, #436, and #476), progesterone treatment increased the cleavage significantly. Cholesterol also reversed the effects o f progesterone effectively (Figure 4.2.). The cleavage rates in control, progesterone, cholesterol, P-cyclodextrin, and combination of progesterone and cholesterol were 23.3 ± 1.7%, 33.3 ± 1.7%, 23.3 ± 1.7%, 20.0 ± 2.9%, 23.3 ± 4.4%, respectively (Figure 4.2.). 103 F I G U R E 4.2. Effects of progesterone and cholesterol on cleavage rates using B O medium (Sperm concentration of 0.2 x 10 6 sperm/ml and in six replicate experiments) • Control • Progesterone S Cholesterol U Cyclodextrine • Progesterone + Cholesterol Bulls with Higher AR Inducibility Bulls with Lower AR Inducibility Sperm Treatments . A total of 600 oocytes were used. (100 oocytes/replicate, 120 oocytes/treatment, and six replicates). . Values were reported as the mean value ± standard errors of the means. .^ Values with different alphabetical superscripts are significantly different. 4.3.4. Progesterone and cholesterol effects on cleavage rate (in T A L P medium) When IVF results were compared for the two groups of bulls (#389, #391, and #461 with more A R inducibility and Bul ls #444, #436, and #476 with a lower A R inducibility), progesterone treatment increased the cleavage rates in both groups. Cholesterol also reversed the effects o f progesterone on cleavage rates in the two groups (Figure 4.4.). The cleavage rates in control, progesterone, cholesterol, P-cyclodextrin, and combination of progesterone and cholesterol were 26.7 ± 1.7%, 50.0 + 2.9%, 21.7 ± 4.4%, 25.0 ± 0.0%, 20.0 ± 2.9%, 104 respectively in the first group with higher A R inducibility (Figure 4.4.) and 18.3 ± 1.7%, 35.0 ± 2.9%, 15.0 ± 2.9%, 16.7 ± 1.7%, 15.0 ± 0.0%, respectively for the second group with lower A R inducibility (Figure 4.4.). F I G U R E 4.3. Effects o f progesterone and cholesterol on cleavage rates using T A L P medium (Sperm concentration of 0.2 x 10 6 sperm/ml and in six replicate experiments) to ed % m U • Control • Progesterone S Cholesterol • Cyclodextrine • Progesterone + Cholesterol Bulls with Higher A R Inducibility Bulls with Lower A R Inducibility . A total o f 600 oocytes were used. (100 oocytes/replicate, 120 oocytes/treatment, and six replicates). L V a l u e s w e r e r epor ted as the m e a n v a l u e ± s tandard errors o f the means . Values with different alphabetical superscripts are significantly different in each group. ( ) . p values in parentheses represent comparison of the related groups with control o f the same group. % When I V F results were compared for all the bulls together, progesterone increased the cleavage rates significantly, and so did cholesterol. The cleavage rates in control, progesterone, cholesterol, P-cyclodextrin, and combination of progesterone and cholesterol were 105 22.5 ± 2.1%, 42.5 ± 3.8%, 18.3 ± 2.8%, 20.8 ± 2.0%, 17.5 + 1.7%, respectively (Figure 4.5.). Progesterone increased the A R percentages by 188.9% (p < 0.05). FIGURE 4.4. Effects o f progesterone and cholesterol on cleavage rates using T A L P medium (sperm concentration of 0.2 x 1 0 6 sperm/ml in 6 replicate experiments) • Control • Progesterone H Cholesterol Q Cyclodextrine • Progesterone + Cholesterol Sperm Pre-Treatments . A total of 600 oocytes were used. (100 oocytes/replicate, 120 oocytes/treatment, and six replicates). . Values were reported as the mean value ± standard errors of the means. •*•. Values with different alphabetical superscripts are significantly different in each group. (). p values in parentheses represent comparison of the related groups with control. 4.3.5. Progesterone and cholesterol effects on zona-binding ability of sperm In the first set of experiments, sperm concentration of 0.2 x 10 6 sperm/ml was used. In this case pre-incubation of sperm with progesterone increased the number of sperm bound to 106 zona pellucida from 11.43 ± 0.6 sperm in control group to 28.93 ± 2 . 1 sperm in progesterone-treated group, which is an increase o f 17.5 sperm bound/oocyte (153.1% increase compared to control group). Sperm treated with both progesterone and cholesterol did not show any increase in the number of sperm bound to the oocyte (Figure 4.5. and Plate 4.7.) F I G U R E 4.5. Effects o f progesterone and cholesterol on number of zona-bound sperm (in three replicate experiments) • Control • Progesterone H Cholesterol • Progesterone + Cholesterol 0.2 x 106 Sperm/ml 5 x 106 Sperm/ml Sperm Treatments . A total of 120 oocytes were used (40 oocytes/replicate, 30 oocytes/treatment, and three replicates). '. Values were reported as the mean value ± standard errors of the means. . Values with different alphabetical superscripts are significantly different in each group. (). p values in parentheses represent comparison of the related groups with control in each group. 107 Plate 4.7. Zona-binding assay with bovine sperm and oocyte (sperm showed by an arrow). A ) Sperm concentration was 0.2 x IO6 B) Sperm concentration was 5 x 106. 108 In the second set o f experiments, sperm concentration of 5 x 10 6 sperm/ml was used. In this group, pre-incubation of sperm with progesterone caused an increase in the number of sperm bound to zona. Progesterone increased the bound sperm from 45.83 ± 2.8 sperm in control group to 73.93 ±3 .7 sperm in the progesterone-treated group, which shows an increase of 28.1 sperm bound/oocyte (61.3% increase compare to control group). Sperm treated with both progesterone and cholesterol, showed no significant increase in the number o f sperm bound to the oocyte. (Figure 4.5., Plate 4.7.). 4.4. DISCUSSION A s documented in previous studies, progesterone induces A R in human spermatozoa, in vitro (Margalioth et al., 1988; Meize l and Turner, 1991; Foresta et al., 1993; Lee et al., 1988). It also induced sperm A R in bovine sperm as observed in the present study. Interestingly, the same concentration o f progesterone as in in vivo (1 u,g/ml) (Osman et al., 1989; Uhler et al., 1992) were used in this study and still an effective increasing effect on A R observed. Similar results were previously reported for progesterone at 1 ng/ml for inducing A R in human sperm (Osman et al., 1989; Blackmore et al., 1990; Tesarik and Mendoza, 1992; Parinaud et al., 1992; Lee et al., 1998). It has been also reported as a steroid secreted by the cumulus oophorus/follicular cells that remain associated with the ovulated zona-enclosed human oocyte (Sabeur et ai, 1996). The positive role of progesterone in inducing the A R of fertilizing sperm in in vivo fertilization has been suggested by several researchers in the past (Kopf and Gerton, 1991; Yanagimachi, 1994). It has been previously reported (Laufer et al., 1984; Zorne et al., 1982; Magalioth et al., 1988; Sueldo et al., 1993) that both zona and progesterone are required for the final fertilization. No effects in hamster egg penetration rates were observed when the medium was supplemented with 10% of follicular phase or preovulatory sera (Magalioth et ai, 109 1988). Similarly luteal sera enhanced egg penetration significantly higher than any other serum. Taken together, progesterone may induce some degrees of A R at the same time that zona pellucida also exerts its A R inducing effects. Progesterone has never been studied in bovine I V F environment, so in the current study, its effcets on I V F system were taken into consideration. When I V F procedures were carried out with the usual sperm concentration for I V F (5 x 10 6 sperm/ml), no significant increase in the percentages of cleavage were observed in effect o f progesterone addition. A s only one sperm finally fertilizes an oocyte, and because progesterone-inducing effects are only partially effective on A R induction, in high concentrations of sperm progesterone effects may be masked by the large number o f sperm. Thus using a lower concentration of sperm/oocyte would probably help us to define the role o f progesterone. When different concentrations of sperm, in the range reported in literature, were studied, it was found that the least possible sperm concentration for I V F was 0.2 x 10 6 sperm/ml. The commonly used sperm concentration in cattle I V F has been reported to vary from 0.5 to 5.0 x 10 6 sperm/ml. Expressed in terms of sperm:oocyte, these would vary from 10.000 to 20,000:1 (Brackett et al, 1982; Bousquet et al, 1984; Lambert et al, 1986; Parrish et al, 1986; L u et al, 1987b). The concentration chosen for this study was 1,000 sperm:oocyte in which acceptable cleavage and blastocyst formation rates were recovered. When 1,000 sperm/oocyte were used in I V F in B O medium, progesterone treated sperm did not increased the rate o f fertilization compared to those untreated sperm. Cholesterol also did not show any effects on fertilzation rates of oocytes. Looking carefully in the conditions o f the experiments, there were some factors that could be responsible for the negative results, caffeine turned out to be the most important one. Caffeine has been classified as a cyclic 110 nucleotide phosphodiesterase inhibitor (Boatman and Bavister, 1988). Caffeine, one of the subgroups o f methyl xanthines, is a (^-adrenergic agonist which increases the level o f c A M P and down stream signal transductions and consequently capacitation and A R (Kopf et al., 1981; Boatman and Bavister, 1984). Pretreatment of rhesus spermatozoa with 1 m M caffeine plus d b - c A M P have been reported to increase the cleavage rates as much as 50% more compared to control (Boatman and Bavister, 1984). Similarly, high levels o f caffeine alone (10 m M ) could produce hyperactive motility in rhesus Spermatozoa (Traub et al., 1982; Boatman and Bavister, 1984). Caffeine treatment o f human spermatozoa in vitro has been also shown to increase the number o f sperm showing irregular swimming trajectories (Gorus et al., 1982; Serres et al., 1982). Moreover, in Japan, N iwa and Ohgoda (1988) reported a synergic effect o f caffeine and heparin in inducing capacitation and A R in frozen-thawed bul l sperm, resulting in increased penetration rates in vitro. Based on these reports, caffeine used in the B O medium could be the masking factor for progesterone effects on A R , as it could induce A R by itself and it was present in all o f the treatments. Thus progesterone could have only an additive effect i f any. Considering the presence of caffeine in B O medium, another medium without caffeine (modified Tyrodes) was used for another sets of IVF experiments. When sperm were pre-treated with progesterone, it increased the cleavage rates by 42.5 ± 3.8% (p < 0.05) compared to control treatment in which the cleavage rate was 22.5 + 2.1%. This further confirmed that the previous experiments did not show the effects o f progesterone because of caffeine. Cholesterol was also shown to be able to inhibit the effects of progesterone on cleavage rates but it could not decrease the cleavage rates to less than control. This was somehow predicted for cholesterol as in A R studies of frozen-thawed bovine sperm also cholesterol-treated sperm did not possess any lower A R than control treatment. The most l ikely explanation for this fact could be due to using frozen semen. In frozen semen, as it has been shown in this study, 111 capacitating is complete right after thawing, when sperm were pre-treated with cholesterol for 10 minutes, in this incubation time cholesterol could not reverse the acrosome-reacted sperm as A R is not a reversible process. Similarly for the proportion of sperm that are capacitated, cholesterol acts to prevent further effects of any A R inducer, so no capacitated sperm would undergo A R in the presence of cholesterol. So the outcome would be the equal percentages of A R in both control and cholesterol-treated groups. For further confirmation of the results in this study, progesterone and cholesterol were added to the frozen-thawed sperm and after required time of incubation, were co-incubated with cumulus-free oocytes. In zona-biding assay, progesterone increased the percentages of cleavage rates significantly. In contrast, cholesterol completely inhibited the effects o f progesterone. This would further confirm the importance of progesterone in I V F systems. Moreover, acrosome-reacted and acrosome-intact spermatozoa have been observed on the zona pellucida (Overstreet and Hembree, 1976) which is again in the favor o f supporting the progesterone inducing effect on fertilization. It has been also reported that preovulatory human follicular f luid enhances the influx o f calcium through the sperm cell membrane (Thomas and Meize l , 1988), the A R percentages (Siiteri et al, 1988; Siegel et al, 1990; Tesarik, 1985; Suarez et al, 1986), and the penetrating capacity in the zona-free hamster ova sperm penetration assay (Siegel et al, 1990; Yee and Cummings, 1988). When lower and higher concentrations of sperm pre-treated with progesterone and cholesterol were compared in terms of zona-binding, the percentages of increase in the zona-bound sperm were much higher in lower sperm concentration. This observation may lead to the possibility o f using progesterone in lower quality sperm. In other words, progesterone was more effective in lower sperm concentrations. Another point was that when sperm were pre-treated with progesterone in I V F experiments, in 5 mi l l ion sperm/ml it did not show any increase in the percentages of cleavage 112 rates while in zona-binding assay the same concentration was shown to increase the number of zona-bound sperm. This could be explained as to be due to partial effects o f progesterone and the fact that from among hundreds of sperm attached to the zona only one would fertilize the oocyte. So in terms of fertilization, using higher sperm concentration doesn't end up with higher cleavage rates but it may cause more sperm to attach to the zona. Sti l l both in zona binding assay and in I V F experiments, the effects o f progesterone and cholesterol were more obvious and larger in lower sperm concentrations. Again this may offer progesterone pre-treatment o f sperm as a practical tool to upgrade the lower quality semen samples to reach the normal level o f fertilization. 5.5. C O N C L U S I O N S Results o f this investigation further strengthen the probability that the frozen-thawed bovine sperm are already capacitated after thawing and have the ability to be induced by progesterone, one of the physiological A R inducers. Further more the observed effective ability o f progesterone to increase cleavage rates in frozen bovine IVF , would further strengthen the possibility of progesterone being utilized to improve the I V F success rate. Lastly it may be concluded that cholesterol inhibitory effects on progesterone-induced cleavage in bovine in vitro fertilization w i l l have the potential to be used in new reversible and effective male contraceptives. There are certain conditions which can be resulted in higher blood cholesterol compare to the normal level and there could be a strong possibility o f sub-normal fertility rates in these patients. Cholesterol inhibitory effects on progesterone showed first in bovine I V F system could explain these kinds of subfertility. Positive results o f adding progesterone to the I V F system would bring a series o f revolutionary possibilities to manipulate I V F system in either bovine or human IVF . 113 CHAPTER 5 FINAL DISCUSSION AND F U T U R E POSSIBILITIES 5.1. S U M M A R Y O F FINDINGS The main purpose of the current thesis was to evaluate the effects o f progesterone and cholesterol on cryo-preserved bovine sperm A R as wel l as in vitro fertilization. Meanwhile the possible relationship between induced A R and in vivo fertility o f bulls was also examined. Moreover, the in vitro effects of different potential A R inducers including 17 P-estradiol, progesterone, db - cAMP, forskolin, and calcium ionophore were studied. The inhibitory effects of cholesterol/p-sitosterol on the induced A R as wel l as the possible mechanism o f cholesterol effects on induced A R were also studied. In the first set o f experiments (Chapter 3), it was found that cryo-preserved bovine sperm were capacitated and could be induced for A R by various known A R inducers such as, progesterone, d b - c A M P , and calcium ionophore. Acrosome reaction induction o f frozen-thawed sperm did not increase significantly with the incubation time up to six hours. Moreover the incubation resulted in a drastic decrease (26.7 ±1.9% decrease) in sperm motility, even after two hours. Although the percentages o f sperm viability remained unaffected, cryopreserved sperm should be used immediately after thawing to retain the maximum sperm motility. Moreover, no significant correlation was found between post-thaw sperm motility and in vivo fertility of the bulls. Similarly, no significant correlation was observed between A R inducibility of sperm and in vivo fertility of the bulls. When cholesterol effect was compared with that of P-sitosterol, the progesterone-induced A R was inhibited by both in the same manner. These results would suggest that 114 cholesterol probably acts by affecting the sperm plasma membrane fluidity and preventing the functional receptor sites to be exposed for ligand-binding. Based on the results o f the first set o f the experiments, it was hypothesized that exogenous progesterone, which serves as an A R inducer, may increases the percentages of cleavage rates in I V F system. Therefore, one can suggest that pretreatment of sperm with progesterone, shortly before their co-incubation with oocytes may support zona-induced A R and consequently increase the fertilization rates o f the oocytes. On the other hand, cholesterol may prevent the effects o f progesterone in I V F system, similar to its inhibitory effects on progesterone-induced A R . Based on this hypothesis, the second set of experiments (Chapter 4) was carried out to elucidate the role o f progesterone and cholesterol in IVF system. Three replicate experiments were carried out with B O medium as a preliminary trial but no significant effect was observed when sperm were pre-treated with progesterone and/or cholesterol compare to control treatment. Lack of effects by steroids could have been due to the sperm concentration used. A s only one sperm fertilizes the oocyte and the effect of progesterone on A R is partial, the chance of the fertilizing spermatozoon to be affected by progesterone would not be very high. Therefore, a range of sperm concentration (0.1-5 mi l l ion sperm/ml) were used for the study, in order to identify the critical sperm concentration that w i l l affect cleavage rates in bovine I V F system. Based on the results, sperm concentration of 0.2 x 10 6 was used to evaluate the effects o f the steroids in bovine I V F system. However, the expected effects o f progesterone and cholesterol were not observed in the six replicate I V F experiments. Therefore, one would suspect that medium supplements such as caffeine might interfere with the steroid effects. T A L P medium was used in the without caffeine in the additional I V F experiments. In these experiments the enhancement effects of progesterone on the percentages of cleavage rates 115 and the blocking effects of cholesterol on progesterone effects were observed. To further confirm the effects of progesterone and cholesterol, sperm-zona binding assay was carried out. The number o f sperm bound to zona pellucida were increased significantly (153% increase) in the presence of progesterone. However, this enhancement effect was totally inhibited by the presence of cholesterol inhibited the effects of progesterone. In summary, this study has demonstrated that cryo-preserved bovine sperm are capacitated immediately after thawing and are ready for A R induction. This may be useful in eliminating the time and effort that are spent for capacitating the sperm prior to A I and IVF . Pre-treatment o f sperm with progesterone was also found to increase the success rate o f bovine I V F system. Inhibition of progesterone effects by cholesterol in bovine I V F system may a potential role o f cholesterol in fertility regulation, in vivo. 5 . 2 . O V E R A L L D I S C U S S I O N Capacitation and A R are not wel l understood in terms of exact mechanisms involved and the factors affecting these processes particularly in cryo-preserved sperm. Numerous attempts have been made to predict bul l fertility by developing various laboratory tests (Saacke, 1982; Den Daas, 1992). However, non o f these tests could predict fertility with acceptable certainty. In the present study it was tried to examine the relationship between induced A R and in vivo fertility o f bulls but no relationship was found between the two. In cryopreserved bovine sperm, A R can be induced by progesterone, d b - c A M P , forskolin, and calcium ionophore. A R induction is inhibited by the presence of cholesterol with the exception o f calcium ionophore. This is perhaps due to different mechanisms of A R induction by different A R inducers. Generally speaking, cholesterol probably exerts its effects v ia changes in the fluidity o f sperm plasma membrane and exposure of surface receptors. Based on the current study, 116 cholesterol may be the stabilizing factor o f sperm plasma membrane during maturation in the epididymis and during ejaculation. Cholesterol can probably prevent the premature A R of sperm in its journey in female genital tract, but it w i l l be removed gradually in the presence o f cholesterol acceptors in vivo. Upon entering the fallopian tubes the cholesterol content of sperm plasma membrane would be probably decreased enough to let the receptors expose and interact with their ligands, such as progesterone and zona glycoproteins. In the last part of this study the objective was to show i f cholesterol and progesterone exert their effects on sperm/oocyte interaction in IVF . Animals were divided into two groups based on their A R inducibility, and were compared for their respective cleavage rates in I V F system with B O medium. Progesterone was shown to increase the percentages of cleavage rates by 42.9% in the group with lower A R inducibility while no effect was found in the group with higher A R inducibility. Similarly when the same experiment was carried out in T A L P medium, progesterone increased the percentages of cleavage rates by 91.3% in the group with higher A R inducibility and by 87.3% in the group with lower A R inducibility. Thus, progesterone may be used to improve fertilization rates in human I V F system, especially those with male factor infertility. The overall cleavage rate was higher in the group with higher A R inducibility. This was consistent with the finding that spermatozoa from oligozoospermic patients were characterized by a reduced in vitro fertilization rate of human oocytes (Matson et al., 1989) and penetration o f hamster oocytes (Aitken et al., 1984). In fact, male variability is l ikely to be a general feature of mammalian I V F (Hillery et al., 1990). How sperm exert control over such embryonic development is not clear but a possible explanation of the phenomenon, based on the results o f the current study can be advanced. Considering the consistent trend of A R inducibility by different A R inducers among individual bulls, the differences among bulls appear to be dependent on their individual semen characteristics. Moreover, between the two 117 groups o f semen samples in this study, those with higher A R inducibility had more fertilization rates in I V F system compared to those with lower A R inducibility. Although in this study, no relationship was found between A R inducibility and non-return rates, but it seems that sperm responsiveness to A R inducers is a highly individual characteristics based on which it would be possible to predict the I V F and A I outcome. Similar view has been taken into consideration by Shi etal. (1991). In terms o f the effects of progesterone and cholesterol on the cleavage rate in I V F system, it was found that progesterone increases the cleavage rates while cholesterol inhibits the effects o f progesterone in IVF system. Although progesterone is not present in the I V F system but in vivo when sperm and oocyte meet, there are two sources available for A R induction prior to fertilization, progesterone and zona pellucida. Thus presence o f cholesterol in such environment would probably decrease the level of A R by inhibiting the progesterone effects and leaving zona proteins as the only source of A R induction. This assumption is further supported with some other reports. It has been reported by Benoff et al. (1993a, 1993b) that the sperm plasma membrane o f four men whose sperm appeared normal yet could not fertilize an egg in vitro contain between two and six times the amount of cholesterol as do the sperm of four men who successfully fathered children through in vitro fertilization (Bennof et al, 1993a, 1993b). A lso men with elevated blood cholesterol concentrations were reported to experience fertility problems and to have elevated concentrations of cholesterol in the plasma membranes o f their ejaculated sperm (Bennof et al, 1993a, 1993b). Benoff et al. (1993a, 1993b) also reported that human spermatozoa whose plasma membranes contain roughly three times the normal concentration of cholesterol have fewer mannose receptors on their heads than do sperm with average cholesterol concentrations. Moreover, it was found that the high cholesterol, low-mannose-receptor sperm have an impaired ability to undergo the acrosomal reaction. 118 5.3. F U T U R E P O S S I B I L I T I E S The results of this study may make a significant contribution to possible applications o f pre-treatment of sperm with different A R inducers to improve the percentages o f cleavage rates in mammalian I V F systems. It remains to be explained why only a small portion of motile viable sperm becomes responsive to A R induction by progesterone, forskolin, and db -cAMP. A lso it would be an important question of relevance that what are the biological implications o f this observation in vitro or in vivo. Moreover, it is possible that a high blood cholesterol may cause sub-normal fertility among individuals as it has been speculated also by Benoff et al. (1993a, 1993b). There are certain conditions, which result in higher blood cholesterol compare to the normal level and may be related to sub-normal fertility rates in humans. Hypercholesterolemia which is an increase in cholesterol 7-ot hyroxylase, a decrease in phospholipid secretions or gallbladder secretions, obesity, high calorie nutrition, a medical history of gallbladder stones, and usage of certain drugs, are conditions which could result in an increase in the level of blood cholesterol as compared to the normal and this may affect the outcome o f individual's fertility. Having a better understanding o f the mechanism of cholesterol effect on A R , would probably further help these patients to have normal fertility. Furthermore, compounds that interfere with the process by which sperm in the epididymis incorporate membrane cholesterol may prove to be useful male contraceptives by inducing premature A R in the spermatozoa. Similarly drugs that prevent the addition of cholesterol may render spermatozoa more prone to undergo a premature acrosomal reaction. In contrast, compounds that prevent the expulsion o f cholesterol during capacitation might also work as contraceptives, as it may happen in Hypercholesterolemia patients to decrease their fertility. A s a support for this hypothesis, several previous studies have shown the relationship 119 of high blood cholesterol and subfertility of human males (Benoff et al, 1993a, 1993b). It remains to be demonstrated i f blood cholesterol levels can serve as efficient agent for fertility regulations in humans. 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Fertil Steril 38:162. 150 Appendix A 1- 1) Ovary Collection Media (1,000 ml): -Pen ic i l l in 100,000 U J - Streptomycin 0.2 gm - Normal Saline (warm to 35.9°C) 1,000 ml 1- 2) Oocyte Aspiration Media (40 ml): - Phosphate Buffered Saline 40 ml - B S A ( 0 . 3 % ) 0.12 gm - Gentamicin(10mg/ml) 200 ul (filter through 0.2 micropore filter) 1-3) Oocyte Maturation Media (10 ml): - Gentamicin (10 mg/ml) 50 u l - T C M - 1 9 9 9.5 ml - F S H (10 mg/ml) 10 u.1 - S O C S (Super Ovulated Cow Serum) 500 u l or 0.5 151 1-4) In Vitro Culture Media (9.5 ml): - Gentamicine (10 mg/ml) 50 u l - T C M - 1 9 9 9 ml - Donor cow Serum 500 ul - Insulin 10 ul 1-5) BO-A Solution (500 ml): - Sodium Chloride 4.3092 gm - Potassium Chloride 0.1974 gm - Calc ium Chloride, Dihydrate 0.2171 gm - Sodium Phosphate, Monobasic 0.0840 gm - Magnesium Chloride Hexahydrate 0.0697 gm - Phenol Red (0.5%) 0.1 ml - Dist i l led Water up to 500 ml 1- 6) BO-B Solution (200 ml): - Sodium Hydrogen Carbonate 2.5873 gm - Phenol Red (0.5%) 0.04 ml - Dist i l led Water up to 200 m l (Pass 5% CO2 into the solution for 30 minutes) 152 1-7) B O Solution (35 ml): - B O - A Solution 26.6 m l - B O - B Solution 8.4 m l - Sodium Pyrovate 0.0048 gm - Gentamicin (10 mg/ml) 35 u l 1-8) Caff-BO Solution (Caffeine BO) (25 ml): - B O Solution 25 ml - Caffeine Sodium Benzoate 0.0243 gm (Sterilized by filtration through a 0.22 um mill ipore filter before use) 1- 9) BO-BSA Solution (10 ml): - B O Solution 10ml - Bovine Serum Albumin 0.06 gm - Heparin Sodium (1000 IU/ml) 36 ul (Sterilized by filtration through a 0.22 urn mill ipore filter before use) 1- 9) Caff-BO-BSA Solution: - Caf f -BO Solution 1 part - B O - B S A Solution 1 part 153 Appendix B DIFFERENT TYPES OF TALP M E D I U M Chemical Name Unit Sp-TALP H-TALP Fert-TALP NaCl m M 100 100 114 KC1 m M 3.1 3.1 3.2 NaHC0 3 m M 25.0 25.0 25.0 NaH2P04 m M 0.3 0.3 0.3 Sodium lactate m M 21.6 21.6 10.0 CaCl2 m M 2.0 2.0 2.0 MgCl2 m M 0.4 0.4 0.5 HEPES m M 10.0 10.0 -Sodium pyruvate m M 1.0 1.0 0.2 BSA mg/ml 6.0 6.0 6.0 Gentamicin Ug/ml 50.0 50.0 50.0 Heparin sulfate ug/ml - 10.0 10.0 154 Appendix C STEPS INVOLVED IN IVF WITH TALP MEDIUM Oocytes aspirated JIU Oocytes matured in vitro in Ham's F-10 plus 10% estrus cow serum (26 h, at 38.5°C, 5% CO2) + Frozen-thawed semen washed with S p - T A L P Fertilization in Fer t -TALP for 15 hours 43-2 x wash in Ham's F-10 plus 10% fetal bovine serum 43-Cleavage in Ham's F-10 plus 10% fetal bovine serum (72 hours) 155 Appendix D STEPS INVOLVED IN IVF WITH BO MEDIUM Oocytes aspirated (Oocyte aspiration media) -43-Oocytes matured in vitro in oocyte maturation media plus 5% estrus cow serum (16 -18h ,a t38 .5°C ,5%C0 2 ) + Frozen-thawed semen washed with C a f f - B O - B S A 43-Fertilization in Caf f -BO-B S A for 16-18 hours 2 x wash in culture media plus 5% super ovulated cow serum 43-Cleavage in culture media plus 5% super ovulated cow serum (48 hours) 156 Appendix E CHEMICAL STRUCTURES Progesterone 157 158 Appendix F PROTOCOL FOR PSA-FITC STAINING (DIRECT IMMUNOFLUORESCENCE ASSAY) 1. After each treatment, place a 5-ul droplet of sperm on multi-spot slides. 2. Spread the droplet uniformly and air-dry the slides. 3. F ix the slides in methanol for 10 minutes. 4. Wash each wel l three times with P B S and 5% B S A . 5. Dilute a 20-mg/ml solution o f F I T C - P S A (1:100) to the final concentration of 20 irg/ml. 6. App ly a 25-u.l droplet of that to the wells and incubate them in a dark chamber for 30 minutes (in a 38.5°C incubator). 7. After incubation remove the excess P S A and wash the slides three times with P B S . 8. Place a droplet o f 80% glycerol in Dulbecco's phosphate buffer saline. 9. Mount each slide with appropriate coverslip and study under the fluorescent microscope (at 400 x to 1,000 x Magnification, excitation filter range of 450-490 nm. 10. Count the sperm on randomly selected fields until 200 spermatozoa are counted from each wel l of slides. 159 Appendix G PROTOCOL FOR 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% B S A . 2. Thaw 1-2 straws of semen and wash in S p - T A L P medium two times. 3. Concentrate and resuspend in H - T A L P or Fer t -TALP medium. 4. Adjust volume to attain a sperm concentration of 0.2 x 10 6/ml or 5 x 10 6 /ml. 5. Draw 20 u l and place in miniplates. add 30 u l to the previous 20 u l to get a total o f 50 u l (at 10 oocytes/drop). 6. Using fine bore pipette, remove cumulus cells from oocytes and add 10 oocytes per drop but do not select oocytes. 7. Co-incubate sperm and oocytes for 4 hours. 8. Pick and move oocytes to next dish. Remove loosely attached sperm by pipeting up and down T E N T I M E S in P B S (you may vary this, but be consistent). 9. F i x the oocytes with 2.5% glutaraldehyde in P B S for 10 minutes. 10. Wash each group of oocytes three times in P B S . 11. Stain with Hoechst 33342 stain for 10 minutes (1 mg/ml in PBS) in dark chamber. Wash again with P B S three times. 12. 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. 13. Keep in a dark, cool place until ready to read in epifluorescence microscope. 14. Use U V - 2 A filter (excitation: 330-380 nm; barrier: 420 nm). 160 Appendix H PRELIMINARY STUDIES F I G U R E H . l . Percentages of A R induced by 30-minute incubation with different concentrations of progesterone (in 18 replicate experiments). J P-Cyclodextrin | | Progesterone Concentration (u,g/ml) . Values were differences between acrosome reaction in control and after each treatment. \ Values were reported as the mean value ± standard errors of the means. ^ Values with different alphabetical superscripts were statistically different (p < 0.05). (). Values in parentheses were the p values for the related group compared to control (p-cyclodextrin). T A B L E H . l The percentages of A R induced by different concentrations o f 17 P-estradiol (in 18 replicate experiments). 17 f3-estradiol s u (0.1 ng/ml) (i ng/ml) (10 ng/ml) % 17 P-estradiol-Induced AR 1.2 ± 0.4% 1.5±0.4%b 3.8 ± 0.4%a . Values were differences between acrosome reaction in control and after each treatment. . Values were reported as the mean value ± standard errors of the means. . Values with different alphabetical superscripts were statistically different (p < 0.05). 161 F I G U R E H . 2 . The percentages of sperm A R increase following addition of progesterone to different concentrations of cholesterol (in 18 replicate experiments). 00 xi U 40 35 30 25 20 15 10 5 0 34.1 3 T 16.8 b T 1.6 C 1.5 C 0.8 C I I I i i—=—i P-Cyclodextrin Progesterone Progesterone Progesterone Progesterone + + + Cholesterol Cholesterol Cholesterol (0.1 ug/ml) (lUg/ml) (10 ng/ml) Sperm Treatments . Values were differences between acrosome reaction in control and after each treatment. \ Values were reported as the mean value ± standard errors of the means. .^ Values with different alphabetical superscripts were statistically different (p < 0.05). . The concentration used was 10 ug/ml. 162 

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