Eastern Illinois University The Keep Masters Theses Student Theses & Publications 1986 The ffecE ts of Various Adenosine and Xanthine Analogues on Mammalian Sperm Motility Daniel Joseph Cushing Eastern Illinois University This research is a product of the graduate program in Zoology at Eastern Illinois University. Find out more about the program. Recommended Citation Cushing, Daniel Joseph, "The Effects of Various Adenosine and Xanthine Analogues on Mammalian Sperm Motility" (1986). Masters Theses. 2726. https://thekeep.eiu.edu/theses/2726 This is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. The Effects of Various Adenosine and Xanthine Analogue s on Mammalian Sperm Motility (TITLE) BY Daniel Joseph Cushing THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 1986 YEAR I HEREBY RECOMMEND THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE 12/L//'lG DATE The Effects of Various Adenosine and Xanthine Analogues on Mammalian Sperm Motility Master's Thesis Presented to the Zoology Faculty Eastern Illinois University In partial fulfillment for the degree Master of Science in Zoology by Daniel Joseph Cushing Fall 1986 12 /l//8'' DATE ABSTRACT Caffeine, theophylline, and a number of other xanthine analogues have been shown to stimulate the motility of mammalian spermatozoa. The mechanism by which these compounds act was assumed to be cAMP phosphodiesterase inhibition. However, it has recently been shown that many of the responses elicited by alkylxanthines and their analogues in various tissues are not the result of cAMP phosphodiesterase inhibition but by antagonism of endogenous adenosine. Using the recently described transmembrane migration method this study observed the effect of a number of xanthine and adenosine analogues on mammalian sperm motility . The hypothe sis to be tested was that the increase in the motility of mammalian sperm is the result of antagonism of adenosine, at the A1 receptor, by alkylxanthine s. Caffeine, theophylline, 1- methylxanthine, 3-methylxanthine and 3-propylxanthine (enprofylline) all increased sperm motility significantly above control. Adenosine also produced a small, but significant, stimulation of motility. cyclohexyladenosine (CHA) , a potent A adenosine receptor 1 -11 - 6 agonist, decreased motility from lo M to l0 M but motility returned to normal as the concentration of CHA - 6 -3 was increased from l0 M to l0 M. Maximum inhibition of -6 motility was produced at l0 M CHA. Theophylline is a known adenosine receptor antagonist, while enprofylline is believed to lack significant adenosine antagonistic properties. Both theophylline and enprofylline reversed - 6 the inhibitory effects of 10 M CHA. It was, therefore, concluded that the alkylxanthine stimulation of mamma lian sperm motility is the result of a mechanism other than adenosine antagonism. i ACKNOWLEDGEMENTS The author wishes to thank Dr. J.R. Lodge and Mr. Robert Becker of the University of Illinois for their assistance with the collect ion and preparat ion of the bovine semen. The author also wishes to thank Dr . Kipp Kruse for his assistance with the presentation of the data and Dr. T. Howard Black for his assistance with the preparation of the chemical structures depicted. A special thanks to Drs . Patrick Docter and William James for their academic and emotional support throughout the duration of this project -- especially the final few weeks. The author is most indebted to Dr . Kip L. McGilliard for being everything that an advisor is supposed be ...and more. To my family, for their constant support, encouragement and love. It would have been impossible to have acc omplished this without ALL of them. Finally, to my mother, who dedicated her life to education and her family . Her love and guidance has, and will always, inspire me to strive for excellence. This thesis is a perfect example of her influence . ii TABLE OF CONTENTS PAGE 1 ACKNOWLEDGEMENTS. • • • . • • • • • . • • . • . • • • • . • . • • . • . LIST OF TA BLES. • . • . • . • iii LIST OF FIGURES .................•..........•••...•• iv DEDICATION. • • . • . • . • • • . vi INTRODUCTION 1 MATER IALS AND METHODS 40 RESULTS 44 Controls ...................................... 44 Effect of xanthine analogues and adenosine ...• 44 CHA Dose-response Analysis 53 Theophylline and enprofylline dose-response analysis . 53 Antagonism of CHA by theophylline and enprofylline ....... 53 DISCUSSION . 64 LITERATURE CITED 72 iii LIST OF TABLES NUMBER TITLE PAGE 1 Various drugs ' effects on mammalian sperm motility. ......................... 9 2 Activity of various compounds at the P-site .................................. 28 3 One-way ANOVA of CHA dose-response a na1 y s i s . 5 6 4 Two -way ANOVA of theophylline-CHA interaction .............................. 57 5 Two -way ANOVA of enprofylline-CHA interaction . .. 58 iv LIST OF FIGURES NUMB ER TITLE PAGE 1 Structure of caffeine ................... 2 2 Structure of theophylline ............... 4 3 Structure of the xanthine core .......... 6 4 Structure of pentoxifylline ............. 10 5 Structure of 2 ' -deoxyadenosine.......... 14 6 Structure of adenosine .................. 18 7 Adenosine receptor array . 21 8 Structures of various adenosine analogues. 2 3 9 Structure of 2' ,5'-dideoxyadenosine ..... 26 10 Structure of xanthine core with respect to adenosine antagonistic properties in most tissues ............................ 31 11 Structure of 1,3-di-n-propyl-8- (2-amino- 4-chlorophenyl)xanthine (PACPX ) ......... 34 12 Structure of 8- [4- [[[[ [2-[ethyl(4- hydroxybenzyl)ammon io]ethyl]amino]­ carbonyl]methyl]oxy]phenyl)-1 ,3-di- n-propylxanthine acetate ................ 36 13 Diagram of transmembrane migration apparatus . ...................... ........ 42 14 Effect of caffeine on human sperm motility ............ .................... 45 15 Effect of theophyl line on bovine sperm motility ................................ 47 16 Effect of 1-methylxanthine and 3-methyl- xanthine on bovine sperm motility.. ..... 49 17 Effect of adenosine on human sperm motility ................................ 51 v LIST OF FIGURES NUMBER TITLE PAGE 6 18 Dose-response of N -cyclohexyladenosine (CHA) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 5 4 19 Dose-re�Ronse of theophy lline alone and with 10 M CHA . 6 0 20 Dose -re��onse of enprofylline alone and with 1 0 M CHA . 6 2 For Ma rgaret Leonce Cushing ( "M()M") Born: 16 February 1934 Died: 20 ()ctober 1986 INTRODUCTION A va riety of tissues has been utilized in the investigation of membrane biochemical events and how such events are manipulated by pharmacologic agents . Although such in vitro studies may not always be directly correlated with to in vivo phenomena , they are often useful predictors of in vivo responses . Sperm cells represent an excellent source of material with wh ich one may study certain membrane phenomena (Babcock et al. 1975) . They are readily available in large quantities and drug effects on motility are quite easily observed. It has been shown that caffeine (1,3,7- trimethylxanthine) (Figure 1), theophylline (3,7- dimethylxanthine) (Figure 2) , 3-isobutyl-1-methylxanthine (IBMX ) and many other xanthine (Figure 3) ana logues have the ability to stimulate the motility of mammalian spermatozoa in vitro (Ame lar et al. 1980; Garbers and Ko pf 1980) . Some wo rkers (Dougherty et al. 1976 ; Multamaki et al. 1980 ; Weeda and Cohen 1982) have shown caffeine to have no effect on motility. However, the general consensus is that these compounds stimulate motility in a dose-dependent manner. A number of other drugs has been employed in the study of sperm mo tility in many mamma lian species . The categories of such drugs include : plasminogen activators Figure 1. Structure of caffeine . 3 :£ � u z z ("fl I 0 z (_) I � ('Y1 C) I u Figure 2. Structure of theophylline . 5 � z z Figure 3. Structure of the xanthine core . 7 � z" mz o __..,. D 8 (Hong et al. 1985a), opiate analogues (Foresta et al. 1985), protein kinases (Schill 1975 ; Schill et al. 1979)' local anaesthetics and beta-blockers (Hong et al. 198lb; Hong and Turner 1982)' mitochondrial respiration inhibitors (Hong and Chiang 1983), psychoactive and other central nervous system (CNS ) acting agents (Cates and Jozefowicz 1970 ; Hong et al. 1982; Thomas and Turner 1983) , va ginal contraceptives (Hong et al. 1983; Louis and Pearson 1985)' natu ra lly occuring diterpenes (Vi jayaraghavan and Hoskins 1985), and hormones (Wang et al . 1983 ; Vasquez et al. 1986) . The responses elicited by these drugs on sperm motility have been catalogued (Table 1) . One other drug that has been evaluated for possible enhancement of sperm motility is l- (5-oxohexyl )-3, 7- R dimethylxanth ine (Pentoxifylline , Trental , Hoechst ) (Figure 4) . Pentoxifylline is a xanthine analogue and as such is expected to inhibit phosphodiesterase and therefore increase the intracellular cyclic adeno sine 3',5 ' -monopho sphate (cAMP ) concentration. This is indeed the mechanism of its action. The resu lt is what wo uld normally be expected-increased sperm mot ility (DeTurner et al . 1978; Apa ricio et al. 1980; Marrama et al. 1985) . The unique aspect of this compound is its ability to work in vivo. The in