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 vivo studies used a regimen of 400mg orally three times per day for six months. These studies are ongoing and mo re data are expected concerning effect on 9
Table 1. Various drugs ' effects on mamma lian sperm motility .
INCREASE DECREASE NO EFFECT
2'-Amino-2- 'deoxy- x adenosine Bicarbonate x Caffeine x cAMP x 2-Ch loroadeno sine x cglorpromazine x N '-Cyclopropyl x carboxamide adenosine D-600 x DAMME x 2'-Deoxyadenosine x 5'-Deoxy-5 '-methyl- thioadensoine x Diazepam x Dihydrotestosterone x Dipyridamole x Estradiol x Fo rskolin x Gossypol x hCG x Histamine x IBMX x Isoprenaline x Kallikrein x Chlordiazepox ide x L�gnocaine x N -Methyladeno sine x 2'-0-Methyladenosine x MnC1 x 2 Naloxone x Nonoxyno l-9 x Pentoxifylline x Phenobarbitone x Phenytoin x Procaine x Progesterone x Propranolol x Stellazine x Streptokinase x Testosterone x Tetracaine x Theophylline x Urokinase x Figure 4. Structure of pen toxifylline . 11
01 I o __ Z - LJ
z I � 0 �
,---... [\J - I (_J ...___..... 0 (_J 01 I (_J 12
motility as well as any adverse effects such as teratogenicity (ability to cause developmenta l malformations) or mutagenicity.
Some workers speculate that xanthines and caffeine in particular may be mutagenic (Joffe 1979 ; Ronen and Marcus
1980; Morris and Weinstein 1981; Aitken et al. 1983) .
Caffeine is a known clastogen (a substance with the ability to break DNA) and since the structure of pentoxifylline is quite similar to that of caffeine their effects on the developing fetus may also be similar
(Weinstein et al. 1972).
A more novel series of compounds available for determination of their effect on sperm motility is the ectokinases (Noland et al . 1986; Sh imomura and Garbers
1986 ). Garbers et al. (1982b) have isolated two classes of peptides from sea urchin eggs that have the ability to stimulate sperm respiration rates and motility. One has been termed "speract" and the other "resact". These proteins have the ability to increase cAMP and cGMP
(cyclic guanosine 3',5'-monoph osphate) concentrations which in turn result in an increase in motility (Shimomura and Garbers 1986).
The most novel theory concerning the mechanism for the stimulation of mamma lian sperm motility is antagonism of endogenous adenosine. Vijayaraghavan and Hoskins
(198 6) examined the effect of various adenosine analogues on the motility of mamma lian spermatozoa . Their findings 13
indicate that adenosine , 2'-deoxyadenosine (Figure 5) and many other adenosine analogues increase cAMP concentrations and ultimately increase motility . They postulated that adenosine is a physiological mo dulator of
sperm mo tility and sugge st that adenosine and various adeno sine analogues elicit their responses via cAMP . This theory is similar to that which will be examined in this the sis.
The mechanism by which xanthines stimulate motility is sti ll the topic of much debate . However , most workers agree tha t an increase in intracellular cAMP is the prima ry determinant (Garbers et al . 197la; Hoskins et al .
1974; Hoskins and Casillas 1975 ; Hoskins et al. 1975;
Levin et al. 1981 ; Tash and Means 1982; Vijayaraghavan et al. 1985) . This fo llows the "second messenger" hypothesis introdu ced by Sutherland (1970 ) where cAMP acts as a second messenger to elicit certain cellular re sponse s.
There are many mechanisms controlling the intracellular concentration of cAMP . The first mechanism involves activity of the enzyme cAMP phosphodiesterase .
Under normal conditions this enzyme is responsible for the conversion of cAMP into 5'-AMP eliminating the second messenger along with the stimu lation that this second messenger elicited .
Xanthine analogues are a class of drugs that has the ab ility to inhibit the activity of cAMP phosphodiesterase . Figure 5. Structure of 2'-deoxyadenosine . 15
D I 16
Caffeine and theophylline have been studied extensively for their effect on sperm motility with via phosphodiesterase inhibition (Garbers et al. 197la ;
Garbers et al. 1971b; Garbers et al . 19 73; Hoskins et al.
1975; DeTurner et al. 197 8; Cheng and Boettcher 1981 ;
Levin et al . 1981 ; Jiang et al . 1984 ; Vijayaraghavan et al . 1985) . These studies leave little doubt that phosphodiesterase inhibition is one possible mechanism responsible for the stimulation of sperm motility by xa nthines.
Another possible factor responsible for altering cAMP ++ is calcium (Ca ) concentration (Garbe rs and Kopf 198 0;
Kopf et al. 1984) . Garbers et al. (1982a) reported that ++ Ca caused an almost 2-fold increase in guinea pig sperm cAMP in the absence of bicarbonate . This would lead one to believe that the motility would also increase . ++ Howeve r, Hong et al. (1985b ) report increased Ca concentration to be detrimental to human sperm motility .
They also found calcium antagonists to increase the mo tility of sperm. There have not been any follow up studies supporting either of these theories. One must therefore view these observations carefully.
Bicarbonate ions may also be responsible for the increase in cAMP as wel l as motility. Garbers et al. ++ (1982a) have shown tha t Ca produced a 25-fold increase - in sperm cAMP concentration in the presence of HC0 . 3 Th is concentration of cAMP is sufficient to elicit an 17
increase the motility of sperm cells (Okamura et al.
1986) . All of these observations led to the conclusion
that cAMP concentration is the primary determi nant
reg ulating sperm motility.
Adenosine (Figure 6) is a known modulator of cellular
responses in a number of tissues (Daly et al. 1981; Daly
1982 ; Dunnwiddie and Fredholm 1984 ; Churchill and
Churchill 1985 ; Daly 1985; Fredholm 1985) . Its roles
include : cardiovascular depression, vasoconstriction in
the kidney , inhibition of platelet aggregation ,
ant ispasmodic and antithrombotic action , stimulation of
steroidogenesis , vasodilation, bronchoconstriction ,
inhibition of neurotransmission , inhibition of insulin
effects , and many more . It is unlike a hormone in that
there is no central site of distribution . Adenos ine is
synthesized and released from most tissues and has direct effects on each specific tissue type . Unlike many
hormones the biological half-life of adenos ine is only a
few seconds . It is not norma lly stored .
Formation of adenosine intracellularly as we ll as extracellularly is primarily from conversion of ATP to ADP
to 5'-AMP and ultimately to adenosine .
Elimination of adenosine occurs mainly by enzymatic
degr adation but also by incorporation into nucleotides .
The enzymes involved in the inactivation of adenosine
include : adenosine deaminase, adenosine kinase, and
adenosylhomocysteinease (Zimmerman et al. 1979 ; Lehninger Figure 6. Structure of adenosine . 19
z�z :r: 0
C\J :r: z z 0 :r: :r: \z__!! u D :r: 20
1982) . These enzymes are present in sufficient quantities
to keep both the extrace llular and intracellular adenosine
concentrations at l-2uM (Daly 1982) .
Adenosine elicits its responses via a complex
receptor array (Daly et al. 1981) (Figure 7) . Adenosine
binds to extracellular and/or intracellular receptors ,
causing a change in the conformation of proteins , altering the activity of adenylate cyclase which in turn controls the formation of cAMP .
Two types of extracellular adenosine receptors have
been identified and studied . They are characterized by their ability to inhi bit or stimu late adenylate cyclase .
The A receptor is a high affinity receptor that , 1 wh en bound by adenosine , functions to inhibit adenylate cyclase (Fain et al. 1972; Trost and Stock 1977; VanCalker et al. 1978; VanCa lker et al . 1979 ; Londos et al. 1980) .
Adenosine has an affinity range of 10-lOOnM for A 1 6 receptors , while some N -substituted analogues are much more potent , having affinities as low as lnM (Daly et al.
1981) . The following is the order of potencies of adenosine analogues for A receptors beginning with the 1 6 6 most potent : N -cyclohexyladenosine (CHA) > L-N - 6 (phenylisopropyl) adenosine (L-PIA) = N -phenyladenosine > 6 2-chloroadenosine = adenosine > N -methyladenosine = 6 benzyladenosine = D-N -(phenylisopropyl)adenosine (D-PIA)
= adenosine 5'-ethylcarboxamide (NECA) (Trost and Stock
1977; Londos et al. 1978) (Figure 8) . Figure 7. Adenosine receptor array (Schwabe 1984). a I Activating Stimulatory adenosine nucleotide receptor (A2) .:;:/�regulatory: . site rc.. ATP Aden,vlate cyclase alvtic. site � cAMP CCL inhibitory
Inhibitory adenosine nucleotide "' Adenosine receptor (A1) ..... :::::-. regulatory site
\11 i 11I l ;; 111;1;!I !: I[iii! lilil !i ii 1:111 , 1:: 1!! ;) t'
I\) I\) Figure 8. Structures of various adenosine analogues. 24
CHJ-...... /CKz-0 �?=? Cl &> -
HOC
HO OH HO OH HO OH 6 CHA PIA 2-chloro N -methyl adenosine adenosine
r-0
a�N &> N
HOCI
HO OH HO OH HO OH 6 6 N -phenyl N -benzyl NECA adenosine adenosine 25
The A receptor response acts in contrast to that of 2 the A (Blume and Foster 1975 ; VanCalker et al. 1978; 1 Bruns 1980) . The A receptor is a stimulatory receptor for 2 adenylate cyclase with an affinity constant in the range of 2-2 0uM (-1000-fold lower affinity than A ). The order 1 of potency of the adenosine analogues at this site are as follows : NECA > 2-chloroadenosine = adenosine > 6 phenyladenosine = N -benzyladenosine > L-PIA = CHA > D-PIA
(Bruns 1980) .
The third site of adenosine action is an intracellular site called the "P-site " (Londos and Wo lff
1977; Stengel and Hanoune 1981) . The P-site is a low affinity site for adenosine binding with affinities on the order of 10-2 0uM . This site is believed to exist either in the cata lytic site of adenylate cyclase or "very near to it" (Stengel 1986) (Figure 5) . However, the P-site is much more structurally demanding than the two extracellular sites . The P-site will tolerate virtually no changes of the purine core (Londos and Wolff 1977; 6 Wo lff et al. 1978) . N -substituted adenosine analogues and alkylxanthines are inactive at this site , wh ile 2- chloroadenosine is only slightly active . The most potent adenosine analogue at the P-site is 2' ,5 ' -dideoxyadenosine
(Figure 9) , which is 10 times more potent than adenosine
(Daly 1982) . Other compounds and their activities at this site are presented in Table 2 (Stengel 1986 ; Stengel et ++ al. 1986 ; Henry et al. 1986) . Manganese (Mn ) seems to Figure 9. Structure of 2',5'-dideoxyadenosine. 27
0 _!Jz :::c \ I z 28
Table 2. Activity of various substances at the P-site .
ACTIVE NON-ACTIVE
Adenosine x
Adenosine arabinoside x
Calcium x
Calmodulin x
2-ch loroadenosine x
CTP x
5'-deoxy-5'-methylthioadenosine x
2'-deoxyadenosine x
Fluoride x
Forskolin x cGMP x
GTP x
Hormones x
Inosine x
ITP x ++ Mn x
Phosphate x
L-PIA x 29
play an important role in the activity of the P-site . In the absence of this ion the P-site is unresponsive to any substance, including adenosine . The reason for this phenomenon is explained in detail elsewhere (Stengel et al. 1986 ; Henry et al. 1986) .
Alkylxanthines have the unique ability to antagonize adenosine at both A and A receptors . Xanthines have 1 2 structural characteristics similar to adenosine and bind in a competitive ma nner to block the effects of endogenous adenosine. This competitive inhibition results in responses that are opposite of those expected for adenosine. For example , antagonism at the A site would 1 result in an increase in the intracellular cAMP concentration, wh ile antagonism at the A site results in 2 a decrease in cAMP .
The above observations have led to the proposal of a new mechanism of action of caffeine , theophylline , and other alkylxanthines . The concentration of alkylxanthine necessary to inhibit phosphodiesterase is much greater than that needed to produce a variety of other pharmacologic effects regulated by adenosine , rendering the phosphodiesterase theory less valid at low concentrations . Using this premise , workers have begun to reevaluate the basic pharmacology of alkylxanthines .
Structure-activity studies have yielded a great deal of information concerning the activity of xanthines at the various adenosine receptor sites (Fredholm 1980; Snyder 30
1981 ; Snyder et al. 1981 ; Fredholm and Persson 1982 ;
Pe rsson 19 82a; Bruns et al. 19 83; Daly et al. 19 83a ; Daly et al. 1983b; Glennon et al. 1984 ; Daly 1985; Daly et al.
198 5; Schwabe et al. 1985; Ukena et al. 19 85; Jacobson et al. 1985; Jacobson et al. 1986; Daly et al. 1986).
Substitution at the 1-position of the xanthine nuc leus appears to be essential for adenosine antagonism wh ile the importance of the 3-position is somewhat controversial (Figure 10). The 3-po sition was proposed by
Pe rsson (19 82) to be unnece ssary for adenosine antagonism.
Howeve r, Ukena et al. (1985) and Schwabe et al. (1985) have shown that 3-n-propylxanthine (enpro fylline) doe s indeed have adenosine antagonistic properties in rat fat and brain cell preparations and platelet aggregation.
The 7- and 9-positions appear to enhance antagonism of adenosine but are less important than either the 1- or the 3-position . Whi le 8-substituted xanthines are the most potent antagonists of adeno sine (Bruns et al. 1983;
Glennon et al. 1984 ; Jacobson et al. 1985; Jacobson et al.
1986; Daly et al. 19 86).
It appears that n-propyl substituent s at the 1- and
3-position are the ideal size for adeno sine antagonism.
Both ethyl and butyl side chains result in a dec rease in potency . Phenyl substituent s at the 8-po sition yield increased potency . Para substituent s on the 8-phenyl group, along with ortho amino groups, add even more specificity (Daly et al. 1986). Figure 10. Structure of xanthine core with respect to adenosine antagonistic properties in most tissues. May increase adenosine antagonism
I 0 I Essential for l adenosine : antagonism !� N N In crease 1 7 '\ adenosine l3 9 antagonism � N 0 N
May increase May increase adenosine adenosine antagonism antagonism
VJ f\) 33
The most potent adeno sine antagonist to date at the
A site is l,3-di-n-propy l-8- ( 2-amino-4-chlorophenyl) 1 xanthine (PACPX) (Bruns et al. 19 83; Schwabe et al. 19 85)
(Figure 11) . Notice that this compound contains all of the substituent s ment ioned above for optima l potency .
PACPX has an affinity constant at A of -o .2nM and is some 1 4 million times mo re potent than xanthine itself and
70,000 times more potent than theophy lline . It is much less potent at the A site, with an affinity of -o .4uM. 2 This difference in affinity is sufficient for studying A 1 receptor phenomena without interference from A effects. 2 One other compound that can be used for A studies is 1 8-[4-[[[[[2-[ethyl (4-hydroxybenzyl)ammonio]ethy l]amino] carbonyl]methyl]oxy]phenyl]-1 ,3-di-n-propylxanthine acetate (Jacobson et al. 19 85) (Figure 12). This compound has an affinity at A of 2.2nM and an affinity at A of 1 2 32 0nM. Even though its affinity for A is 10-fold less 1 than PACPX it exhibits an A :A binding ratio of 145 which 1 2 makes it ideal for studies involving effects of A 1 antagonism.
The best A antagonists ava ilable at the present time 2 are : l-n-propyl-3,7-dimethylxanthine , l-ethyl-3-methyl- xanthine and 8-[4- [ (carboxymethyl)oxy]phenyl]-1,3-di-n- propylxanthine . Their A :A affinity ratios are 3, 3 and 2 1 2 re spectively . These are not nearly sufficient for distinguishing A -mediated phenomena from A -mediated 2 1 effects. Development of a specific A antagonist wou ld be 2 Figure 11. Structure of 1,3-di-n-propyl-8-(2-arnino-4- chlorophenyl)xanthine (PACPX) . 35
r-f u
(\J zI z z
o __" z-£ z--/. £ I \\ � (\J 0 I :r: u U (\J :r: u (T') I u Figure 12. Structure of 8-[4-[[([[2-[ethyl(4-hydroxy benzyl)arrunonio]ethyl]amino]carbonyl]methyl]oxy]phenyl]- 1,3-di-n-propylxanthine acetate. 37
I 0
u • <( 0 I
-- z
:C' (Tl
z- u
z-� I � r-..... 0 � u 38
a major accomplishment and would certainly lead to a better understanding of this system.
As one can see , there is an overwhelming amount of ' evidence linking the function of adenylate cyc lase in many tissue s with adenosine receptor ac tivity. The existence of adenosine receptors in mature mamma lian sperm cells has not yet been established . However , it has been reported that radiolabe lled adenosine analogues bind to rat spermatids (Murphy and Snyder 1981; Murphy et al. 1983) and human and bovine sperm adenylate cyclases are known to be inhibited by high levels of adenosine (Hyne and Lopata
1982; Brown and Casillas 1984) . These observations illustrate the need for further investigation of mature mammalian sperm cells for possible effects of adenosine and its analogues on motility .
There have been no structure-activity relationships presented with respect to the role of xanthine analogues nor adenosine analogues in the control of mamma lian sperm motility . This thesis wi ll provide a structure-activity relationship of some xanthine and adenosine analogues and also expound on the various mechanisms which control mammalian sperm motility.
This study wi ll test the effect of selective A 1 receptor agonists on mamma lian sperm motility and the modulation of such effects by various xanthine analogues.
The hypothesis to be tested is that the increase in the motility of mamma lian sperm is the result of antagonism of 39
adeno sine , at the extracellular A adenosine receptor , by 1 alkylxanthines. MATERIALS AND METHODS
Caffeine , theophylline , 1-methylxanthine , 3- 6 methylxanthine, adenosine and N -cyclohexyladenosine were purcha sed from Sigma Chemical Co. (St . Louis, MO , USA) .
3-n-propylxanthine (enprofylline) was a gift from AB Draco
Inc . (Astra , Sweede n) .
Fresh human semen samples were obtained by masturbation from healthy volunteers and were allowed to liquefy at room temperature for 30 minutes prior to investigation. Fresh bovine samp les were collected by artificial vagina and allowed to liquefy for 1 hour prior to investigation. Total sperm counts were made of all samples prior to inve stigation and were within normal limits.
All drugs were dissolved in phosphate buffered saline at a pH 7.3 to the desired concentration . Each semen sample wa s divided into lOOul aliquots and then mixed with
50ul of the appropriate drug solution. A 2:1 semen-buffer mixture wa s used as a control . -6 For agonist/antagonist assays l0 M CHA (determined
from dose-response analyses as the optimal motility inhibiting dosage) was combined with various concentrations of the desired alkylxanthine .
Drug influence on motility was determined using the transmembrane migration method (TMMM) introduced by Hong et al . (1981) . A lOOul aliquot of the semen-drug mi xture 41
was pipetted into the hollow plunger of a 2rnl Sabre syringe (Sabre Intnl.) (Figure 13) . A sheet of polycarbonate membrane (Nuclepore Ltd.) , 13mm diametre with evenly spaced Sum pores , was bonded to the lower end of th e plunger with methy lene chloride . The upper chamber was then placed into a lower chamber , which consisted of a lOml glass vial containing 2ml of phosphate buffered saline. The height of the upper chamber was adjusted using a cork stopper until the fluid levels of the two chambers were even. This apparatus was then incubated in a Ha ake Dl-L (Haake Buchler Inc.) circulating water bath ° at 37 c for 2 hours .
At the end of the 2 hour incubation period the upper chamber wa s removed and the sperms in the lower chamber were killed by the addition of SOul of 10% formalin solution. A one drop sample was taken from both chambers and counted using a haemocytometre . The sperms able to cross the membrane were termed moti le . The percentage of motile sperms was calculated and used as the indicator of drug effects .
Statistical analysis was accomplished using a paired t-test for single dose studies, one -way analysis of variance (ANOVA) with the mu lticomparison tests of Tukey and Dunnett along with two-way analysis of variance for dose-response studies (Sokal and Rohlf 1981) .
Significance was determined at the 0.01 critical level. Figure 13. Diagram of transmembrane migration apparatus. 43
SEMEN PHOSPHATE BUFFER RESULTS
Controls
Control sperm motility values using the TMMM (Hong et al. 1981) were in the range of 60% to 80%. The mean of the controls was 67.5%. These values are somewhat greater than those of other workers but nonetheless were consistent throughout th is study . The tota l sperm counts of bovine samples ranged from 400 to 600 million ce lls/ml while human subjects ranged from 60 to 80 million cells/ml. These are within norma l limits and were consistent throughout the study.
Effect of Xanthine Analogues and Adenosine
Pilot studies we re performed to determine if the data gathered in this study using the TMMM were consistent with those of other workers using the same method . Caffeine
(Figure 14) and theophylline (Figure 15) (lmM) each caused increases of approximate ly 10% and 30% in sperm moti lity , respectively. These resu lts were statistically significant (P < 0.01) and in agreement with previously reported values using this , and other, methods .
Both 1-methylxanth ine and 3-methylxanthine (lmM) each significantly (P < 0.01) increased bovine sperm motility by approximat ley 25% (Figure 16) , while adenosine (lmM) caused a small (5%) but significant (P < 0.01) increase in human sperm motility (Figure 17). The increase elicited Figure 14. Effect of caffeine on human sperm motility. Bars represent +/- SEM of four determinations. (*) indicate observations statistically significant from control (P < 0. 01; t-test). 46
w _J z 0 H a: w I- LL Z LL 0 <{ u u
*
0 0 0 0 0 0 0 m ro " lD U1 ""
31110� % Figure 15. Effect of theophylline on bovine sperm motility. Bars represent +/- SEM of four determinations. (*) indicate observations statistically significant from control (P < 0.01; t-test). w z H _J _J >-J 0 I a: 0... I- 0 z w 0 I u I-
0 0 0 0 0 0 0 m co " tO U1 �
31Il0V'J % Figure 16. Effect of 1-methylxanthine and 3-methyl xanthine on bovine sperm motility. Bars represent +/- SEM of four determinations. (*) indicate observations statistically significant from control (P < 0.0 1; t-test). 50
L 2: E E -M -M
w w z z H H :r: :r: I- I- z z <( <( x x _J _J _J >- >- 0 :r: :r: er: I- I- r- w w z :2: L 0 I I ..,.; u (T') lZZJ � f\S'i
0 0 0 0 0 m co ...... lO LO
31I10V'J % Figure 17. Effect of adenosine on human sperm motility. Bars represent +/- SEM of four determinations. (*) indicate observations statistically significant from control (P < 0. 01; t-test). 52
w z _J H 0 u a: 0 I- z z w 0 0 u * 0 0 0 0 0 O'l CD " lO ID 31110� % 53 by adenosine was consistent with other recent data (Hoskins and Vijayaraghavan 19 86) . CHA Dose-response Analy sis The results show that CHA causes a dose-dependent -11 -6 decrease in human sperm moti lity from 10 M to 10 M (Figure 18) . This decrease was significant (P < 0.01) (Table 3) . In contrast , at co ncentrations ranging from -6 -3 l0 M to 10 M CHA caused the mot ility to return to the control value . This observation was also significant (P < 0.01) . Theophy lline and Enprofy lline Dose-response Analyisis Theophylline dose-response analysis was observed -7 -3 within a concentration range of 10 M to 10 M. The results show that theophylline significantly (P < 0.01) (Table 4) increased human sperm motility abo ve control. -7 -3 Wi thin a concentration range of l0 M to 10 M enprofylline also caused a significant (P < 0.01) (Table 5) concentration-dependent increase in human sperm mo tility at a lower dose than that caused by theophylline in this assay . An tagonism of CHA _ey Theophy lline and Enprofylline Various concentrations of theophylline were -6 administered in combination with 1 x l0 M CHA to test the abi lity of theophylline to antagonize CHA. The data show 6 Figure 18. Dose-response analysis of N -cyclohexyl- adenosine (CHA). Bars represent +/ SEM of six determinations. Buffer control value = 69. 6 (+/- 2.3). (*) represents observations significantly different from control (P < 0.01; Dunnett). 55 (T') ' � ' lf) I - w L .µ tD 1 . ,...... ,....; " U) ...... I w ,....; 0 E CD - I CJ> 0 0 -.:""4 ,....; I -.:""4 ..... I N ..... 0 0 0 0 0 0 I m CD ...... lO lf) � =i1r10� % Table 3. One-way ANOVA of CHA dose-response analysis. *** ANOVA TABLE *** SOURCE OF SS MS F p DOSE 9 7291.5 810.17 14.02 <.00 1 ERROR 62 3581.75 57.77 TOTAL 71 10873.25 57 Table 4. Two-way ANOVA of theophylline-CHA interaction . *** ANOVA TABLE *** SOURCE DF SS MS F p DRUG 1 2634.16 2634.16 116.94 <.001 DOSE 5 3317.56 663.51 29.46 <.001 INTERACTION 5 1574.59 314.92 13.98 <.001 ERROR 60 1351.56 22.53 TOTAL 71 8877.88 Table 5. Two-way ANOVA of enprofylline-CHA interaction . *** ANOVA TABLE *** SOURCE DF SS MS F p DRUG 1 1093 .84 1093.84 89 .2 <.001 DOSE 5 11025.63 2205.13 179.83 <.001 INTERACTION 5 24 50.00 49 0.00 39 .96 <.001 ERROR 60 735.75 12.26 TOTAL 71 15305.22 59 -7 tha t theophylline , above l0 M, reverses the inhibition of human sperm motility induced by CHA (Figure 19). -6 Once again using l0 M CHA as the agonist , a dose- response analysis was obtained for the effect of enprofylline on CHA-induced inhibition of motility. The data obtained indicate that enprofylline has the ability to reverse the inhibition induced by CHA and to stimulate -? human sperm motility in concentrations above 10 M (Figure 2 0) • Fig�re 19. Dose-response of theophylline alone and with 10 M CHA . Bars represent +/- SEM of six determinations . (*) represents observations significantly different than theophylline alone (P < 0. 01; Tukey-Kramer) . Contro l value for theophylline alone = 69.7 (+/- 1. 2). Control value for theophylline with CHA = 45.95 (+/- 3. 1). 61 ...--.. :£ (T) :J I '<:""i w z I -- a u _J w w z H (!) _J I _J >- I Q_ 0 w I t'-. I- I CJ) 0 r-f CD I 0 0 0 0 0 Ol CD t'-. (!) LO :31110� % 20. Figire Dose-response of enprofylline alone and with 10 M CHA. Bars represent +/- SEM of six determinations. (*) represents observations significantly different than enprofylline alone (P < 0.01; Tukey-Kramer). Control value for enprofylline alone = 64.98 (+/- 1.2). Control value for enprofylline with CHA = 40.25 (+/- 1.6). 90 * * * 80 * w _J 70 H � 0 L 60 � • ENPROFYLLINE ALONE • ENPROFYLLINE + CHA ( 1uM) 50 40 -+-��-.--��..--��.--���----.��----, -8 -7 -6 -5 -4 -3 °' log ENPROFYLLINE (moles/litre) w 64 DISCUSSION A number of methods have been employed in the evaluation of drug effects on sperm motility . Such methods include photomicrography (Mak ler et al. 1980) t spectrophotometry (Atherton 1979) and turbitometric analysis (Levin et al 1980) . These methods are costly and complicated . Therefore , the need for an inexpensive , uncomplicated , yet accurate method for evaluating the effects of various compounds on sperm motility is certainly apparent. One method fulfilling all of these requirements is the transmembrane migration method (TMMM) developed by Hong et al. (1981) . Comparing sperm counts by TMMM with those of direct microscopic examination yielded a correlation coefficient of r= 0.9613. TMMM consistently overestimated the motility compared to direct microscopic observation. The authors stated that this may be due to the difference in temperature between these two methods. ° Specimens in the TMMM were incubated at 37 c, wh ile the others were not incubated. The control values reported in the present study are somewhat higher than those reported by other workers (Hong et al. 1981) . However , these values were consistent within the study . The increase in human sperm motility induced by caffeine wa s similar to that reported by other workers using the same method (Hong et al. 1981) . It was , therefore , concluded that the method was sound. In recent years there have been numerous reports outlining the effects of alkylxanthine analogues on sperm motility (Garbers and Kopf 1980) . These studies show a positive correlation between alkylxanthine concentration and sperm moti lity . The findings of the present study are consistent with those of previous work showing theophylline and caffeine to significantly increase manunalian sperm motility above control. The action of cAMP appears to be direct ly dependent on alkylxanthine concentration. The increases in bovine and human sperm moti lity produced by lmM theophylline and in human sperm motility produ ced by lmM caffeine can be attributed to inhibition of cAMP phosphodiesterase . The premise for such a conc lusion lies in previous reports in which caffeine and theophylline were shown to inhibit sperm phosphodiesterase and to increase cAMP at these concentrations (Garbers et al . 197la ; Vijayaraghavan et al. 1985) . The effects of 1-methylxanthine and 3-methylxanthine on sperm motility had not previous ly been investigated . The results showed a significant increase in bovine sperm moti lity associated with introduction of these compounds . The mechanism by wh ich these compounds stimu late motility is most likely inhibition of phosphodiesterase . Since all alkylxanthines are thought to have the characteristic of 66 inhibiting phosphodiesterase it is likely that such effects would be observed in this assay . A purine uptake system could be responsible for the observed increase in motility; however, this possibility has not fully been established (Hoskins 1986; personal communication) . Comparison of the effects of 1-methylxanthine and 3- methylxanthine should prove helpful in discerning a structure-activity relationship for alky lxanthines with respect to sperm motility . There was no significant difference in the strength of the responses elicited by either 1-methylxanthine compared with 3-methylxanthine . It was, therefore, concluded that ,while either 1- substitution or 3-substitution of the xanthine core enhanced sperm motility, neither of the two is required for the enhancement of sperm motility Adenosine caused a sma ll but significant increase in human sperm motility . This result is in agreement with that of Vijayaraghavan and Hoskins (1986) who reported a dose-dependent increase in bovine sperm moti lity associated with adenosine within a concentration range of -6 -4 10 M to l0 M. The mechanism by which adenosine acts is still unclear. It may be the result of binding to an extracellular adenosine receptor, to an intracellular adenosine receptor (P site) , or via a purine uptake system (Vijayaraghavan and Hoskins 1986) . 6 Unlike adenosine, N -cyclohexyladenosine (CHA) , a potent A adenosine receptor agonist, did not cause an 1 67 increase in motility above control. CHA caused a concentration-dependent decrease in sperm moti lity between -11 -6 -S lo M and l0 M which returned to normal between 10 M -3 -6 and l0 M. Maximum inhibition was observed at l0 M. A complete dose-response ana ly�is of CHA had not previous ly been reported; however, one report (Vijayaraghavan and Hoskins 1986) found CHA to be "ineffective in stimu lating motility ". This statement is consistent with the results obtained here , where an increase in human sperm motility was not observed . The mechanism of action can be selected from the same premises as the action of adenosine itself. The precise mechanism by which inhibition of motility and return to norma l sperm mo1.ility are controlled is still unc lear. One li kely possibility is that CHA in lower concentrations binds primari ly to A receptors causing 1 decreased moti lity . At higher concentrations CHA may also bind to A receptors, opposing the effects mediated by A 2 1 receptors . Theophylline alone caused a dose-dependent increase in the motility of human sperm within a concentration -7 -3 range of l0 M to l0 M. It appears (Figure 18) that if one were to increase the concentration further , motility would increase accordingly . This result is consistent with that of Jiang et al. (1984) who reported theophylline to increase sperm motility in a dose-dependent manner from -4 -2 l0 M to l0 M. Likewise enpro fy lline alone caused a dose-dependent 68 increase in human sperm motility within a concentration -7 -3 range of 10 M to 10 M. This finding has not previously been reported. It appears that enprofylline is more potent than theophylline in this assay since the -6 stimulation of motility was initiated at 10 M whereas theophylline stimulation of motility began at -4 concentrations above 10 M. This is consistent with other reports where enprofy lline is five times more potent than theophylline as a bronchodi lator and cardiovascular stimulant (Persson 1982b; Persson and Erjefalt 1982 ; Persson et al 1983) . The mechanism of action for both theophy lline alone and enprofylline alone is probab ly phosphodiesterase inhibition since theophy lline is known to inhibit phosphodiesterase at 10-4M and enp rofylline is known to inhibit this phosphodiesterase at concentrations as low as -6 10 M. Theophyl line wa s ab le to reverse the inhibition of -7 mot ility induced by CHA at concentrations above 10 M. This reversal may be attributed to antagonism of adenosine at the A adenosine receptor. 1 Support for this concept is that theophy lline has a binding affinity at A receptors (in oth er tissues) of 1 -6 10 M, which is in the range of the theophylline reversal of CHA effects in this assay (Daly et al. 1981; Schwabe et al. 1985; Daly et al. 1986) . Another alternative is that theophylline may be inhibiting phosphodiesterase. This possibility is less valid in this assay since the concentration needed to inhibit phosphodiesterase is in the millimolar range and theophylline reverses the moti lity inhibition of CHA in the micromo lar range . One other theory is that theophylline is interacting with a purine uptake system and inhibiting the effect of CHA . This is merely speculative since there is no evidence that such a system is operating in sperm . Enprofyl line, like theophylline, also reversed the -6 inhibitory effect on moti lity of l0 M CHA. At -7 concentrations above l0 M enprofylline caused a significant increase in motility. Recall that at these same concentrations enprofylline alone stimulated the motility of human sperm. That result was attributed to phosphodiesterase inhibition . Keeping this in mind the mechanism of action for the reversal of CHA inhibition by enprofylline is, at least in part, due to phosphodiesterase inhibition . It is also generally believed that enpro fylline lacks any adenosine antagonistic properties (Persson 1982a; Fredholm and Persson 1982; Persson et al. 1986) . There fore, one may rule out competitive antagonism of CHA at the A adenosine receptor as the cause for increased 1 sperm motility. However, some workers have associated enpro fylline with antagonism of adenosine (Schwabe et al. 1985; Ukena et al. 1985) . Ukena et al. (19 85) reported that enprofyl line is mo re likely to bind to A receptors 2 than A receptors . This supports the conclusion of this 1 70 thesis that the effect of enprofylline is not due to antagonism at the A receptor . 1 If, as the previous reports suggest, enprofylline were binding to A receptors, then sperm motility should 2 decrease as the result of A receptor antagonism by 2 enprofylline . However, since enprofylline caused an increase in sperm motility it could not have antagonized A receptor mediated effects as this theory implied. 2 These results, along with the fact that enprofylline is a potent phosphodiesterase inhibitor , also support the conclusion that enprofylline is acting by a mechanism other than adenosine receptor antagonism. The lack of adenosine receptor modulation of sperm motility may be explained by the recent theory that mature sperm lack the functional guanine regulatory subunit (Stengel 1986) . This theory draws support from the fact that NaF and cholera toxin are unable to activate the adenylate cyclase of mature ram sperm. Still another possible mechanism is that the increase in cAMP, causing the increase in sperm motility, is secondary to an increase in calcium concentration (Vijayaraghavan and Hoskins 1986) . Calcium is known to increase the levels of cAMP in other tissues (Dunwiddie 1984) . Therefore, since alkylxanthines cause an increase in calcium concentration, it may be that the increase in sperm cAMP is secondary to the alkylxanth ine-stimulated increase of calcium levels. 71 The data presented in this thesis , as we ll as those of other workers , show that mo tility is stimulated by compounds having one characteristic in common -- all are known to increase cAMP levels. However , since adenosine effectively inc reased mo tility at lmM and CHA caused variable concentration-dependent effects on motility , it seems logical to assume that there must be some type of adenosine mo dulation of sperm motility. If alkylxanthines and adenosine an alogues do indeed act through an external adenosine receptor array , the structure-activity relationship of such action must be different from that observed in many other tissues . It has been shown that enpro fylline and theophylline have the ab ility to stimulate the motility of human sperm . 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