motility activation, respiratory stimulation, and alteration of ca2+ transport in bovine sperm...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 221, No. 1, February 15, pp. 291~303.1983

Motility Activation, Respiratory Stimulation, and Alteration of Ca*+ Transport in Bovine Sperm Treated with Amine Local

Anesthetics and Calcium Transport Antagonists’

JAI PAL SINGH,2 DONNER F. BABCOCK,3 AND HENRY A. LARDY4

Institute for Enzyme Research and Dqmrtment of Biachemistty, Univendy of Wiscansin, 1710 University Avenue, Madison, Wisconsin 53706

Received October 7, 1982

Optimal concentrations of dibucaine and other structurally related tertiary amines, variously classified as local anesthetics, Ca” transport antagonists or calmodulin-directed agents greatly stimulate respiration and the motility of bovine spermatozoa in a reversible manner. Because dibucaine also increases lactate production by sperm made dependent upon glycolysis, the induced metabolic stimulation is probably a secondary response to the greater energy demands resulting from increased motility. Microscopic and time lapse photomicrographic examinations indicate that dibucaine increases the proportion of motile cells and alters the predominant linear swimming path to a peculiar figure eight pattern of movement. Frame by frame analysis of video recordings indicate that this pattern of movement closely resembles, or is identical to the characteristic “motility activation” that occurs during the capacitation sequence which obligatorily precedes fertilization of many, if not all, mammalian species. Di- bucaine and the Ca2+ transport antagonists, D6OCl and TMB-8, inhibit the net uptake of Ca2+ by sperm suspensions. The dose-response relationships indicate that inhibition of Ca2’ uptake does not bear a causal relationship to the activation of motility and metabolism and further suggest a common action of these agents rather than selective effects of D600 and TMB-8 upon Ca2+ channels in the sperm plasma membrane. In addition, dibucaine and D606 each induce release of that Ca2+ which was accumulated by intact sperm in a preliminary incubation in the absence of the drugs and also inhibit uptake of Ca2+ by digitonin-treated sperm. Apparently, therefore, local anesthetics have a direct deleterious action on sperm mitochondrial function. Treatment with the high concentrations of local anesthetics that are required to inhibit uptake of Ca2’ com- pletely results in a rapid and irreversible immobilization of the sperm. This loss of motility is either not mediated, or mediated indirectly, through an action of the drug on mitochondrial function because sperm similarly become immotile when a glycolytic substrate is supplied simultaneously.

’ Supported by Grant AM10334 from the National Institutes of Health.

Mammalian spermatozoa undergo var-

* Present address: Collaborative Research, Inc., ious morphological and functional alter-

Main St., Waltham, Mass. 02154. ations during their passage through the

* Present address: Department of Clinical Pathol- male and female reproductive tracts. For

ogy, Oregon Health Sciences University, Portland, example, sperm acquire motile compe- Ore. 97291. tence during transit from the caput to the

’ Author to whom all correspondence should be ad- cauda epididymis (l), although they re- dressed. main quiescent until initiation of motility

291 ooO3-9861/83/030291-13$03.00/O CoPYright 0 1983 h.v Academic Press. Inc. All riuhta of reproduction in any form reserved.

292 SINGH, BABCOCK, AND LARDY

occurs upon ejaculation or upon dilution of the epididymal contents in vitro. A further alteration of motility, known as activation, takes place during sperm capacitation in vivo (2) or in vitro (3-5). The mechanisms underlying these matura- tional processes and their significance for the ability of sperm to fertilize eggs are not known.

The components of a CAMP-mediated regulatory system are present in both invertebrate and mammalian sperm (6, 7). Pharmacological treatment with phosphodiesterase inhibitors or CAMP derivatives stimulates motility and metabolism of intact sperm (8,9) and CAMP apparently enhances the flagellar beat of demembranated sperm whose motility is restored by the addition of Me and ATP (10-13). Al- though the targets of CAMP action have not been identified, these results suggest that CAMP-mediated regulation is exerted rather directly upon the contractile ele- ments of the sperm flagella.

Some evidence also suggests that Ca2+ has a regulatory role in the control of sperm motility. Extracellular Ca2+ strongly affects motility of intact hamster (14,15), mouse (15), and rat (16) sperm and limited entry of extracellular Ca, induced by ionophore A23187 (17), stimulates motility of guinea pig (3), bovine (17), hamster (18), ram (19), and sea cucumber (20) sperm. Entry of extracellular Ca2+ also is associated with motility activation and the acrosome reaction of sperm of several species during capacitation in vitro (3,21,22). Although direct addition of Ca2+ alters the motility of reactivated ciliary and flagellar axonemes of various protozoa (23-26) and of invertebrate sperm (27, 23) only inhibitory effects of Ca2’ on the motility of demembranated reactivated mammalian sperm have been reported (11, 13). How- ever, both invertebrate (29) and mammalian (11, 30, 31) sperm contain large amounts of the calmodulin protein that mediates the action of Ca2+ in many other cells (32). Recently calmodulin has been localized in the ciliary axonema of Tet- ruhymena (33) and in guinea pig (34), bovine (35), and canine (11) sperm flagella where it might be expected to have a stim-

ulatory action upon dynein ATPase as has been demonstrated in vitro (36).

The local anesthetics have profound effects upon a wide variety of biological systems, either initiating or inhibiting secre- tory (3, 21, 37), contractile (38, 39), and metabolic (40-43) responses and processes associated with cell division (44, 45). Be- cause Ca2+ mediates most, if not all, of these responses, demonstration of a stimulatory action of local anesthetics upon mammalian sperm motility further supports an involvement of Ca2+ in control of the flagellar contractile apparatus.

MATERIALS AND METHODS

Bovine epididymides were a gift from Oscar Mayer

and Company (Madison, Wise.) obtained within 1 h of slaughter. Epididymal spermatozoa were collected

and washed as described previously (17). The standard medium employed (medium NKM) consisted of

110 mM NaCl, 5 rnr.r KCl, 10 mM sodium morpholinopropane sulfonate, pH 7.4. Chemicals were ob-

tained from standard commercial sources except for the following generous gifts: o-isopropyl-cu-(N-

methyl-N-homoveratryl)-y-aminopropyl)-3,4,5-tri- methoxyphenylacetonitrile hydrochloride (D600), Knoll A. G., Ludwigshafen, Germany; A23187, Eli

Lilly, Indianapolis, Indiana; 8(N,iV,diethylamino)-oc- tyl-3,4,5-trimethoxybenzoate (TMB-8), Dr. Richard

D. Feinman and Dr. Thomas C. Detwiler, State Uni-

versity of New York, Downstate Medical Center, Brooklyn, New York; trifluoperazine, Smith, Kline and

French, Philadelphia, Pennsylvania. Respiratory measurements were performed in a

Gilson Oxygraph (Gilson Medical Electronics, Mid- dleton, Wise.) equipped with a Clark oxygen electrode. Washed sperm (l-l.5 X 10’ cells) were equil-

ibrated at 3O”C, then transferred to the electrode chamber containing buffer, metabolic substrates, and pharmacological agents to give a total volume of ei-

ther 1.5 or 3.9 ml. In experiments that employed digitonin to increase

membrane premeability selectively, washed cells were resuspended in Medium MSM consisting of 250 mM

mannitol and 70 mM sucrose that were deionized, then made 20 mM in sodium morpholinopropane sulfonate, and 1 mM in sodium phosphate, pH 7.4. Preliminary

experiments established that treatment with 100 pg digitonin/lO* cells for 5 min was sufficient to produce maximal respiratory stimulation in the presence of

5 mM sodium succinate (46), to induce release of the cytosolic enzyme, phosphoglucose-isomerase (determined by a spectrophotometric assay that coupled the released activity to added glucose 6-phosphate dehydrogenase, Ref. (47)). and to abolish extrami-

LOCAL ANESTHETICS ACTIVATE SPERM 293

tochondrial uptake of Caz’ induced by high doses of

A23187 (17).

Uptake of %a’+ by sperm suspensions was determined by the filtration technique previously used in

this laboratory (17, 46). To determine release of accumulated Ca*+, sperm (l-2 X lo* cells/ml) were in-

cubated in medium NKM containing 10 mM b-hydroxybutyrate and 0.25 mM CaClz that was labeled

with ‘%a*’ (2500 cpm/nmol). After 30 min at 3O”C, cells were collected by centrifugation (1000~ for 1 min) and resuspended in the same medium without

Ca’+. After appropriate addition of drugs or chelating agents, or both, aliquots (0.3 ml) were removed, again subjected to centrifugation, and radioactivity in the

supernatant was determined by techniques previ-

ously used (17, 21, 46). Net uptake and release of Ca” by sperm suspen-

sions were monitored by observation of the optical absorbance of the metallochromic indicator antipyr-

ylazo III at ‘790 and 720 nm (48) in an Aminco DW2 spectrophotometer (American Instrument Co., Silver

Springs, Md.). Photomicrographic determinations of motility were

performed as described previously (17) with the generous cooperation and assistance of Elaine Water- man and Dr. John Sullivan of American Breeders’

Service (DeForest, Wise.). In some experiments, the presence or absence of motility of the activated state

induced by local anesthetics was judged subjectively by simple microscopic examination. Video micro-

graphic analyses were performed on dilute sperm suspensions (10’ cells/ml) in medium NKM contain-

ing 2 mg bovine serum albumin (Fraction V, Sigma)/ ml. The suspensions were examined in either open

droplets or in the specialized counting chambers employed in the photomicrographic studies. The Sony

Betamax video recorder was interfaced to a low light camera (MT1 65, Dage MTI, Inc., Michigan City, Ind.),

and a digital event marker (Panasonic WJSOO). Mo- tility patterns were reproduced by tracing the sperm location from the video monitor display in 30-40 con-

secutive video frames. Lactate formation was determined by assay (49) of

perchloric acid extracts prepared after 10 min of in-

cubation of sperm suspensions in medium NKM containing 10 mM fructose.

RESULTS

Eflects of dibucaine on sperm respiration. Previous publications from this (8, 9, 50) and other (see Ref. (7) for review) lab- oratories showed that caffeine and related phosphodiesterase inhibitors stimulate the respiratory activity of the sperm of various mammalian species. Comparison of Figs. 1A and C shows that optimal con-

81 ImM OIBUCAINE

FIG. 1. Respiratory stimulation induced by dibu-

Caine and by caffeine. 1 ml of washed epididymal sperm (IO-l.5 X lo9 cells) were incubated at 30°C for

5 min, then transferred to the oxygraph chamber containing 2.9 ml of medium NKM plus 10 mM /3-

hydroxyhutyrate. After a linear respiratory rate was

established, caffeine or dibucaine (0.1 ml) was added to the indicated final concentration. In tracing B,

@-hydroxybutyrate was deleted. In tracing D car- honylcyanide p - trifluoromethoxyphenylhydrazone

(FCCP) was also added. Calculated respiratory rates

(rg atoms (O/108 cells X h) are shown parentheti- cally.

centrations of either caffeine or dibucaine increased the rate of oxidation of an ex- ogenous metabolic substrate by bovine epididymal sperm to a similar extent. In the absence of added oxidative substrate (Fig. lB), respiration declined to the basal rate within 5-10 min after addition of dibu- Caine. It was noted also that preliminary incubation in the absence of substrate at 37°C for 30 min almost abolished the response to the subsequent addition of dibucaine (data not shown). Together these results suggest that the sperm acetylcar- nitine pool is important to the respiratory response to dibucaine, as it is to the response to caffeine (50).

Respiratory stimulation followed the addition of caffeine by l-3 min (Fig. 1C) and was sustained until anaerobiosis was achieved at all concentrations tested (l-20 mM). Figures 1A and D show that the respiratory response to dibucaine was im- mediate, but transient at higher drug concentrations. These higher doses of dibu- Caine apparently damage mitochondrial function because respiratory inhibition was not reversed by the protonophore carbonylcyanide - p - trifluoromethoxy - phenylhydrazone or by caffeine.

The dose-response curve for initial re-


0, DIBUCAINE (mM)

FIG. 2 Respiratory stimulation by dibucaine and the subsequent addition of caffeine. Washed sperm (1.0 X 10’ cells) were incubated as in Fig. 1. After a

added to the indicated final concentration and the total volume adjusted to 3.9 ml. The respiratory rate observed in the succeeding 2- to 3-min interval was recorded (A) along with the rate observed after the subsequent addition of 3 my caffeine (D).

spiratory stimulation by dibucaine (Fig. 2) shows that the concentration (0.6 mM) required for half-maximal activation of sperm oxidative metabolism (the AD& is similar to that required to elicit pharmacological responses in other biological systems (see Ref. (51) for review). The ef- fective dose was lower at higher pH (data not shown), probably due to increased dif- fusion of the active, uncharged form of the drug into the cells (the pK, for dibucaine is 7.8). Figure 2 also shows that the respiratory rsponse to the combination of dibucaine and caffeine does not exceed that produced by optimal concentrations of dibucaine alone and suggests a common rate- limiting step in the metabolic responses to the drugs.

Evaluutim of pharmucological activity of related amine anesthxtics. Several structurally related anesthetics were tested for their stimulatory action on sperm respiration (Table I). Although lipid partition coefficients are not available for all of the compounds, the most active agent tested, TMB-8 (52,53), is highly lipophilic whereas compounds with low lipid solubility (54, 55) such as procaine and benzocaine, were ineffective. The active compounds also contain a tertiary nitrogen whereas the

secondary amine, benzocaine, was inac- tive. Although more information is required to characterize fully the chemical features that are required for activity, it may be concluded that the observed stimulatory effects are not the result of general membrane perturbation because n-bu- tanol, a widely used perturbant, was in- active at concentrations up to 10 mM.

Eflects of local anesthetics and cue&e cm sperw~ motility. Subjective evaluation of motility indicated that both caffeine and local anesthetics induce vigorous sperm movement. In control preparations, less than 20% of the sperm exhibited vibrational tail movement and little or no pro- gressive motion was apparent in the motility chamber. Caffeine initiated flagellar movement in 60-70s of the sperm, many of which showed linear progression. Di- bucaine (1 mM) induced flagellar beating in a similar proportion of the sperm. How- ever, the pattern of movement was altered markedly. In the presence of concentrations of dibucaine, D600 or trifluoperazine that induce maximal respiration, the flagella beat with a whiplash motion, and, although some sperm swam progressively with large amplitude displacement of the head about the mean swimming path, most of the cells moved in an apparent figure eight pattern. In time-lapse photomicro- graphs (Fig. 3) these patterns of movement are readily distinguished. Progres- sive movement was recorded as a series of “tracks” left by the path of the asymmet- rical head of the sperm. Sperm swimming in a figure eight path produced a circular or pinwheel pattern. In addition many sperm revolved rapidly about a center cre- ated by attachment of the sperm head to the surface of the slide. (In subsequent experiments, inclusion of albumin in the incubation media largely prevented this attachment.) Frame by frame analysis of video records reveals that the figure eight swimming pattern observed in real time examination is, in fact, more accurately described as circular swimming with large amplitude displacement of the sperm head from the mean swimming path. Tracings of the position of the sperm head in con- secutive video frames over an approximate

295 LOCAL ANESTHETICS ACTIVATE SPERM

TABLE I

STRUCTURE AND ACTIVITY OF CAFFEINE AND VARIOUS ANESTHETIC COMPOUNDS ON SPERM RESPIRATION

CAFFEINE

TRIFLUDPERAZlNE

DIBUCAINE

BUTACAINE

F’ROCAINE

BENZDCAINE

0.2

0.4

FN FHs H&O F-KH,),-N-(CH,l, 0.5

OCH, CHKH,),

H2N

0.6

0.9

H2t+(=J- COOCH2CJ12-N<$ B IO

n+Q-Cooc2H, .5

Note. Dose response relationships were determined as in Fig. 2 and used to calculate the concentrations of the agents required for half-maximal activation of respiration. Values shown (except for procaine and henzocaine) are the mean of two to three determinations that did not differ by more than 10%.

time interval of 1 s contrast the typical circular pattern observed for cells treated with dibucaine (Fig. 4A) to the linear pattern observed for the majority of caffeine- treated sperm (Fig. 4B). Sperm treated with optimal concentrations of D606 or trifluoperazine also display circular swimming behavior. At higher concentrations of trifluoperazine, the sperm flagella become severely bent or looped or both, and the cells rapidly become immotile.

Full traces, over a smaller time interval, of the head and flagella of sperm treated with dibucaine (Fig. 4C) are highly similar to those which originally documented motility activation (2).

Relationship between stimulation of mx~

tility and metabolic activity. After treating sperm with digitonin (Table II), sperm motility ceased but respiratory activity of the exposed sperm mitochondria was briefly accelerated, returned to a rate comparable to that of intact sperm, then slowly declined over a period of l-3 min. Addition of the protonophore, ClCCP, to intact sperm stimulated respiration fourfold, but comparable addition to digitonin-treated sperm, during the period when respiration had returned to the basal rate, resulted in no enhancement of respiratory activity. Treatment with /3-hydroxybutyrate and digitonin resulted in a prolonged (>5 min) increase in respiratory activity and the exposed mitochondria remained responsive


FIG. 3. Actions of caffeine, dibucaine, and D600 on sperm motility assessed by photomicroscopy. Washed epididymal sperm (1 X 10s cells/ml) were incubated in medium NKM plus 10 rnrd /3-hy-

droxybutyrate for 5 min at 30°C. Motility was recorded by the photomicroscopic technique described under Materials and Methods after the following additions: A, none; B, 3 rnrd caffeine; C, 1 mM

dibucaine; D, 1 mM D600. The exposure time employed was 2 s.

to ClCCP. Thus @hydroxybutyrate effectively replaced endogenous cytosolic substrates that are required for accelerated respiratory activity. Most importantly, however, treatment with dibucaine did not stimulate the respiration of exposed sperm mitochondria that were supported by /3- hydroxybutyrate.

An indirect stimulatory action of dibu- Caine on respiratory activity of intact sperm is also indicated by the observation (Table III) that dibucaine increased motility and metabolism of intact sperm that were made dependent upon glycolysis by treatment with the respiratory inhibitor, antimycin A. Comparable actions of caffeine on both glycolysis and respiration were previously explained as a probable secondary consequence of the increased energy demands that result from enhanced motility (9). We conclude that dibucaine likewise has its primary stimulatory action on sperm motility.

Reversal of dibucaine action and lack of dependence upon extracellular Ca”‘. In many systems the physiological effects of amine anesthetics are reversed by high concentrations of Ca2+ (51). Addition of Ca2+ in concentrations up to 10 mM did not diminish the stimulatory effects of dibu- Caine upon motility and respiration of bovine sperm (data not shown). However, the respiratory activity of sperm did return to the basal rate following removal of dibu- Caine by gentle washing (Fig. 5A). The whiplash pattern of motility was also lost, but vibrational tail movement persisted in 50-60s of the cells. Both motility and respiration were restimulated by a second exposure to dibucaine. In controls that were continuously exposed to dibucaine during the washing procedure, motility and respiration remained elevated (Fig. 5B).

Inhibiticm of Cat+ Uptake and its relationship to kinetic and metabolic stimulation by local anesthetics. Previous studies

LOCAL ANESTHETICS ACTIVATE SPERM

FIG. 4. Actions of local anesthetics and caffeine on sperm motility assessed by analysis of video recordings. Washed epididymal sperm (1 X lo7 cells/ml) were incubated in medium NKM containing

10 mM @-hydroxybutyrate for 5 min at 25°C. Tracings of representative portions of video records were made as described under Materials and Methods. (A and C) Sperm treated with 1 mM dibucaine. (B) Sperm treated with 7.5 mM caffeine.

(1’7, 46) established that oxidative metabolism supports the accumulation of Ca2+ by the mitochondria of intact bovine spermatozoa. Figure 6 shows that whereas caffeine at concentrations up to 2.5 mM did not affect the extent of accumulation of Ca2’, TMB-8, D600, and dibucaine were inhibitory (IDm = 0.6, 1.3, 1.1 mM, respec- tively). Because the concentration required for 50% inhibition of Ca2’ uptake is in each case considerably more than the ADr,,, for stimulation of respiration (Table I), inhibition of Ca2+ uptake apparently is not part of the mechanism that activates motility and respiration.

Although we cannot exclude a possible action of the transport antagonists on the permeability of sperm plasma membranes to Ca2’, studies with digitonin-treated cells (Table II) indicate that dibucaine exerts a direct inhibitory effect on uptake of Ca2+. Endogenous mitochondrial substrates support a limited amount of up-

take in the exposed sperm mitochondria that was complete by the end of the 5-min observation period of these experiments. If @-hydroxybutyrate was also supplied, uptake increased nearly threefold during this initial period (and continued for pe- riods of up to 15 min). Dibucaine (1 mM) resulted in a comparable inhibition of uptake (70%) in intact and digitonin-treated cells. D600 (1 mM) had equivalent effects on intact sperm and sperm made perme- able with digitonin.

Release of accumulated Ca” by dibu- Caine. Figure ‘7 shows the washout, under various conditions, of 45Ca2t from sperm that had accumulated the isotope in a preliminary incubation. In the absence of further treatment the half-time for release was approximately 2 h. In the presence of EGTA,5 to prevent reuptake of label,

‘EGTA = Ethyleneglycol - bis(p - amino - ethyl ether)-N,N’tetraacetic acid.

TABL

E II

EFFE

CT

OF

DIBU

CAIN

E AN

D D6

00

ON

RESP

IRAT

ION,

M

OTI

LITY

, AN

D Ca

l’ UP

TAKE

IN

IN

TACT

AN

D DI

GIT

ONI

N-TR

EATE

D SP

ERM

Inta

ct

Digi

toni

n-tre

ated

Cond

itions

Resp

iratio

n

(jig

atom

s 0

108

cells

X

h)

Ca*’

upta

ke

(nm

ol

Ca*+

10s

cells

) M

otilit

y

Resp

iratio

n

(pg

atom

s 0

l@

cells

X

h)

Car’

upta

ke

(nm

ol

Ca2+

108

cells

) L2

M

otilit

y

Endo

geno

us

subs

trate

Endo

geno

us

subs

trate

+ CI

CCP

(2.5

J&

M)

5 rnM

b-

Hydr

oxy

butyr

ate

5 mM

&H

ydro

xy

butyr

ate

+ Cl

CCP

(2.5

PM

)

5 rnM

/3-

Hydr

oxy

butyr

ate

+ di

buca

ine

(1

mM

)

5 m

y @

-Hyd

roxy

bu

tyrat

e

+D60

0 (1

m

rd)

0.37

+

0.05

1.60

-+

0.0

5

0.37

f

0.07

2.40

+

0.30

0.86

Et

0.1

0

0.80

f

0.11

7.9

+ 1.

4 Pr

esen

t 0.

37

+ 0.

08

8.8

f 3.

0 Ab

sent

m

$

- Ab

sent

0.

42

+ 0.

06

- Ab

sent

8

7.5

+ 1.

2 Pr

esen

t 0.

65

+ 0.

25

21.2

f

1.6

Abse

nt

P

0.1

+ 0.

03

Abse

nt

1.31

f

0.43

0.

1 f

0.03

Ab

sent

2

2.7

* 0.

4 Ac

tivat

ed

0.36

f

0.09

6.

2 f

1.7

Abse

nt

F

1.0

* 0.

5 Ac

tivat

ed

0.41

+

0.10

3.

5 -c

1.

4 Ab

sent

Note

. W

ashe

d sp

erm

(3

-5

X 10

s ce

lls)

were

in

cuba

ted

for

5 m

in

at

30°C

in

m

ediu

m

MSM

in

the

pres

ence

or

ab

senc

e of

di

gito

nin

and

the

indi

cate

d ad

ditio

ns

of

fl-hyd

roxy

butyr

ate.

Af

ter

linea

r re

pirat

ory

rate

s we

re

esta

blish

ed,

CICC

P,

nupe

rcai

ne,

or

D660

we

re

adde

d as

in

dica

ted.

Fo

r de

term

inat

ion

of

Ca*’

upta

ke,

para

llel

prel

imin

ary

incu

batio

ns

were

co

nduc

ted

in

the

pres

ence

of

0.

08

mb(

an

tipyr

ylaso

III

an

d di

huca

ine

D6OO

or

CI

CCP

wher

e in

dica

ted.

Up

take

wa

s ob

serv

ed

in

the

L-m

in

perio

d fo

llowi

ng

addi

tion

of

0.2

mM

CaCI

z. M

otilit

y wa

s ob

serv

ed

micr

osco

pica

lly

by

rem

oval

of

aliq

uots

at

th

e en

d of

ea

ch

expe

rimen

t. Va

lues

sh

own

are

the

mea

n f

SEM

(n

=

3).

LOCAL ANESTHETICS ACTIVATE SPERM

TABLE III

STIMULATION OF SPERM GLYCOLYSIS AND MOTILITY BY DIBUCAINE AND CAFFEINE

299

Additions

Aerobic incubation Anaerobic incubation’

Lactate formation Lactate formation (nmol/(108 cells X min)) Motility (nmol/(lOs cells X min)) Motility

None 0.017 f 0.003 Present 0.12 f 0.02 Present

1 mM Dibucaine 0.13 f 0.014 Activated 0.30 f 0.04 Activated

3 mM Caffeine 0.033 + 0.004 Activated 0.26 + 0.03 Activated

Note. Washed epididymal sperm (10 X 10s cells) were incubated for 3 min in 4 ml of medium NKM containing 10 mM glucose before the indicated additions. After 10 min at 30°C motility was observed microscopically

and aliquots were removed for determination of lactate production as described under Materials and Methods. Values shown are the mean +- SEM (n = 3).

a Anaerobic incubation conditions were simulated by treatment with the respiratory inhibitor, antimycin

A (5 PM).

washout was accelerated two- to threefold. Rapid and complete release of accumulated Ca” was induced within l-2 min after addition of ionophore A23187. A slower and less extensive release was also induced by 1 mM dibucaine. However, in other experiments (not shown here) net release of

FIG. 5. Reversible nature of the respiratory stim-

ulation induced by dibucaine. Washed sperm (1.5 X 10’ cells) were incubated and respiratory rates,

before and after the addition of 1 mM dibucaine were determined as in Fig. 1. Sperm were then collected by sedimentation (2OOOg/min), resuspended in an

equal volume of either (A) medium NKM plus 10 mM @-hydroxybutyrate, or (B) medium NKM plus 10 mM p-hydroxybutyrate and 1 mM dibucaine. The proce-

dure was repeated and the suspensions returned to the oxygraph chamber. The calculated respiratory

rates ?,pg atoms/lO* cells X h) are shown parenthet- ically.

endogenous mitochondrial Ca2+ was not induced by addition of 1 mM dibucaine to cells that were subjected to a preliminary incubation without Ca”. Moreover, motil-

1 I I I

0.5 1.0 1.5 2.0 CONCENTRATUN ht.4,

FIG. 6. Inhibition of Ca2+ uptake by local anes-

thetics and Ca’+ transport antagonists. Washed ep-

ididymal sperm (l-l.2 X lo8 cells/ml) were incubated in medium NKM containing 10 ml p-hydroxybutyr-

ate, 0.5 mM sodium phosphate, 0.2 mH CaClc labeled with ‘%a*’ (1000 cpm/nmol) and the indicated concentrations of drugs; l , caffeine; A, TMB-8; n , D600;

0, dibucaine. After 15 min at 3O”C, triplicate aliquots of 50 ~1 were removed for determination of radioiso- tope uptake. Values shown are the mean + SEM (n

= 3).

300 SINGH, BABCOCK. AND LARDY

01 ’ I 0 5 IO IS 20 25 30 35

TIME (mid

FIG. ‘7. Release of accumulated Caa+ induced by ion-

ophore A23187 and by dibueaine. Washed epididymal sperm (5 X lo9 cells) accumulated %a’+ during a

preliminary incubation as described under Materials

and Methods. After collection by centrifugation, sperm were resuspended at 3 X 108 cells/ml of the same medium without Cae+. Further additions were: 0, none;

0, 1 mM EGTA at 0 min; A, 1 PM A23187 at 15 min; n , 1 mM dibucaine at 15 min. Aliquots were removed

at the indicated times for determination of ‘%a’+ release as described under Materials and Methods.

ity activation was still observed upon addition of dibucaine to cells that were first depleted of endogenous mitochondrial stores by treatment with A23187. It is unlikely, therefore, that release of mitochondrial Ca2+ by local anesthetics is required for activation of motility by these agents.

It was also observed that the decline in motility induced by concentrations of dibucaine exceeding 1 mM occurred with a time course that paralleled release of endogenous stores of divalent cations, suggesting that deleterious actions on mitochondrial function were responsible for loss of motility. However, it was found subsequently that glycolytic substrates were incapable of sustaining motility in the presence of 2 mM dibucaine. Because 1.7 mM dibucaine induces lysis of eryth- rocytes (55), it is probable that nonspecific

damage to sperm membranes also occurs at these concentrations. Preliminary experiments indicate that whereas 2 mM dibucaine causes rapid release of the acro- somal enzyme hyaluronidase, 1 mM dibu- Caine is ineffective, suggesting that membrane integrity indeed may be adversely affected at the higher concentration.

DISCUSSION

Garbers et al. (8) first reported that motility and metabolism of bovine epididy- ma1 spermatozoa are stimulated by treatment with caffeine or other methylxan- thines. Similar effects were noted later with sperm from other mammalian species (14, 56, 57). Because addition of ex- ogenous cyclic nucleotide derivatives or of inhibitors of cyclic nucleotide phosphodiesterase produce similar effects and because these inhibitors induce a moderate increase in sperm CAMP content (8), it was postulated that an increase in intracellular CAMP concentrations initiates a sequence of events that leads to enhanced activity of the flagellar machinery. The nature of these hypothetical events remains to be discovered.

A preliminary study by Garbers (58) also indicated that dibucaine stimulated kinetic and metabolic activity of bovine sperm. In contrast, inhibitory actions of various local anesthetics on sperm motility were noted in previous publications from this (21) and other (11, 59, 60) lab- oratories.

It was found here that optimal concentrations of dibucaine and the structurally related compounds D600, TMB-8, and trifluoperazine all have comparable stimulatory actions on sperm motility and metabolism. Moreover, the present study rec- onciles earlier disparate observations by showing that higher concentrations of local anesthetics depress sperm respiratory and kinetic activity. Similar concentration-dependent stimulatory and inhibitory actions of local anesthetics have been observed in other cellular systems (38, 51).

It is shown here that, as with caffeine (9, 50), the respiratory stimulation produced by local anesthetics is apparently a


secondary consequence of the greater energy demands resulting from increased kinetic activity and also probably requires acetylation of the sperm carnitine pool. However, caffeine and dibucaine do not have identical actions on cellular function in sperm. Although both agents increase the proportion of motile cells, the swimming behavior in the presence of dibucaine or D600 is characterized by whiplash motion of the flagella and apparent figure eight pattern of movement. Similar behavior is observed during capacitation of hamster (3, 4), guinea pig (4), and mouse (5) sperm, suggesting that local anesthetics induce alterations of motility that re- semble or are identical with the physio- logically important process termed motility activation.

In contrast, the swimming path of epididymal sperm is much less affected by treatment with caffeine. The increase in the proportion of motile cells produced by treatment with caffeine may have its physiological counterpart in the initiation of motility that occurs at ejaculation. In- deed, some evidence indicates regulation of this process by cyclic AMP (11-14, 57, 61-63).

Cyclic AMP-dependent protein kinase activity, assayed in cellular extracts, is not increased by preliminary treatment of sperm suspensions with local anesthetics. Therefore, it is likely that the stimulatory action of these agents upon sperm motility and in metabolism is not mediated by cyclic AMP (64). Instead a separate mechanism, probably mediated by Ca’+, apparently regulates motility activation. This hypoth- esis is consistent with previous demon- strations of net uptake of Ca2+ during capacitation in vitro (21, 22, 65) and of increased kinetic and metabolic activity induced by treatment of bovine sperm with ionophore A23187 and Ca2+ (17). It was also found that dibucaine mobilizes Ca2+ from extramitochondrial stores and thereby increases apparent free intracellular Ca2+ concentrations (64) consistent with reported mobilization, by local anesthetics, of Ca2+ from the inner surface of the red cell membrane (66). Thus considerable experimental evidence supports the postu-

lated roles of intracellular Ca2+ concentrations as a positive effector of sperm motility activation and as a mediator of local anesthetic action.

Local anesthetics, Ca2+ transport antagonists, and the calmodulin-directed agent, trifluoperazine, all appear to have similar inhibitory effects on cellular functions at concentrations greater than those required for stimulation of sperm motility and metabolism. Multiple mechanisms may underlie these inhibitory actions. Some previous studies (67) but not others (68) detected an inhibitory effect of local anesthetics upon Ca2+ uptake and electron transport activity (43) for mitochondria isolated from various sources.

The inhibition of Ca2+ uptake into exposed sperm mitochondria by dibucaine and D600 (shown here) may explain the ability of these drugs to inhibit Ca2+ uptake into intact bovine sperm, of D600 (69) and of verapamil (70) to inhibit uptake of Ca2+ into sea urchin sperm and of verapamil to inhibit in vitro fertilization with mouse gametes (71). An action of D600 on mitochondrial function has not been noted previously.

D600 and verapamil effectively reduce cardiac contractility at lop6 M by inhibiting entry of Ca2+ through voltage-sensitive “slow Na+ channels” (72). Complete inhibition of the voltage-sensitive Ca2+ channels of nerve requires lop4 M D600 (73). At these concentrations, D600 also inhib- its “fast Na+ channels” in both cultured cardiac and nerve cells (74). Thus, although an additional action of D600 upon Ca2+ transport across the sperm plasma membrane cannot be excluded, available experimental evidence does not constitute pharmacological characterization of pu- tative sperm plasma membrane Ca2+ channels.

It was also reported (75) that 1O-4 M D600 blocked an increase in cyclic AMP content of guinea pig sperm that was induced by exposure to extracellular Ca2’ and that was suggested as a mediating event in the sperm acrosome reaction. In contrast, we found (65) that under very similar conditions, D600, like dibucaine (21), enhances uptake of Ca2+ and the acrosome reaction


of these cells. An explanation for this dis- crepancy is not apparent. However, de- polarization of guinea pig sperm membranes induced by K+ and monitored by cyanine dye fluorescence measurements, does not include the sperm acrosome reaction (‘76), also suggesting that the influx of Ca” required for this exocytotic event does not occur through voltage-sensitive channels.

It was demonstrated that a variety of local anesthetics interfere with the action of calmodulin in vitro (77, 78). However, the action of Ca2+ (and presumably of calmodulin) is not required for reactivation of motility in detergent-treated sperm (11, 13, 27, 28) so that calmodulin probably plays a modulating rather than obligatory regulatory role in sperm motility. Thus it is unlikely that interference in calmodulin’s action accounts for the complete inhibition of motility that follows treatment of intact sperm with high concentrations of local anesthetics. Disruption of mitochondrial function also seems incapable of explaining motility inhibition because provision of a glycolytic substrate does not prevent or alleviate the action of these drugs. Instead loss of membrane integrity probably accounts for inhibition of motility observed at high drug concentrations.

ACKNOWLEDGMENTS

We thank Dr. John Sullivan for photomicroscopic

motility determinations and Dr. Julius Adler for use of video micrographic equipment.

REFERENCES

1. BEDFORD, J. M. (1975) in Handbook of Physiology

(Greep, R. O., and A&wood, E. G., eds.), Sec- tion ‘7, Vol. V, pp. 303-317, Amer. Physiol. Sot.,

Washington, D. C. 2. YANAGAMACHI, R. (1970) J. Reprod Fed 23,193-

196. 3. YANAGAMACHI, R. (1975) Bid Reprod 13, 519-

526.

4. KATZ, D. F., YANAGAYACHI, R., AND DRESDNER, R. D. (1978) J. Z&prod Fert 52, 167-172.

5. FRASER, L. R. (1977) J. Exp. ZooL 202.439-444. 6. GARBERS, D. L., WATKINS, H. E., TUBB, D. J., AND

KOPF, G. S. (1978) Advan. C&ic Nucl Res. 9. 583-595.

7. HOSKINS, D. D., AND CASILLAS, E. R. (1975) in

Advances in Sex Hormone Research (Singhal,

R. L., and Thomas, J. A., eds.), pp. 283-324, University Park Press, Baltimore, Md.

8. GARBERS, D. L., LUST, W. D., FIRST, N. L., AND

LARDY, H. A. (1971) Biocktiq 10,1825-1831. 9. GARBERS, D. L., FIRST, N. L., AND LARDY, H. A.

(1973) Biol Reprod 3, 589-598.

10. LINDEMANN, C. B. (1978) CeU 13, 9-18. 11. TASH, J. S., AND MEANS, A. R. (1982) Biol Reprod

26, 745-763. 12. MORISAWA, M., AND OKUNO. M. (1982) Nature

(London) 295,703-704. 13. MOHRI, H., AND YANAGAMACHI, R. (1980) Exp. Cell

Res. 127, 191-196.

14. MORTON, B., HARRIGAN-LUM, J., ALBAGLI, L., AND

Jooss, T. (1974) Biochem Biophys Res Com- mun 56, 372-379.

15. MORTON, B. E., SAGADRACA, R., AND FRASER, C. (1978) Fed. Sted 29, 695-698.

16. DAVIS, B. K. (1978) Proe. Sot Exp Bid Med 157,

54-56.

17. BABCOCK, D. F., FIRST, N. L., AND LARDY, H. A. (1976) J. Biol Chem 251,3881+X86.

18. LUI, C. W., AND MEIZEL, S. (1979) J. Exp. ZOOL 207.173-186.

19. SHANS-BORHAN, G., AND HARRISON, R. A. P. (1981)

Gamete Res. 4,407-432.

20. INOUE, S., AND TILNEY, L. G. (1982) J. CeU BioL 93, 812-819.

21. SINGH, J. P., BABCOCK, D. F., AND LARDY, H. A.

(1978) Bioch.em. J. 172,549-556. 22. TRIANA, L. R., BABCOCK, D. F., LORMN, S. P., FIN,

N. L., AND LARDY, H. A. (1980) Biol Reprod

23,47-59. 23. ECKERT, R. (1972) Science 176,473-481.

24. NAITOH, Y., AND KANEKO, H. (1972) Science 176, 523-524.

25. HYAMS, J., AND BORISY, G. (1978) J. CeU Sci 33, 235-253.

26. BESSEN, M., FAY, R. B., AND WITMAN, G. B. (1980) J. Cell Biol 36, 446-455.

27. BROKAW, C. J. (1979) J. CeU. Bid 82, 401-411.

28. GIBBONS, B. H., AND GIBBONS, I. R. (1980) .I. CeU Bid 84, 13-27.

29. GARBERS, D. L., HANSBROUGH, J. R., RADANY, E. W., HYNE, R. V., AND KOPF, G. S. (1980) J.

Reprod Fert. 59,377-381. 30. BROOKS, J. C., AND SIEGEL, F. C. (1973) Bicde-m

Biophgs. Rea Commun 55,710-716.

31. JONES, H. P., BRADFORD, M. M., MCRORIE, R. A., AND CORMIER, M. J. (1978) Bicwhem Biophgs. Res. Commu% 82,X%4-1272.

32. MEANS, A. R., AND DEDMAN, J. R. (1980) Nature (L.fmdim) 286, 73-77.

33. JAMIESON, G. A., VANAMAN, T. C., AND BLUM, J. J. (1979) Proc Nat. Ad Sci USA 76,6471- 6475.

34. JONES, H. P., LENZ. R. W., PALEXITZ, B. A., AND


WORMIER, M. J. (1980) Proc Nat. Ad Sci USA 77,2772-2776.

35. FEINBERG, J., WEINMAN, J., WEINMAN, S., WALSH,

M. P., HARRICANE, M. C., GABRION, J., AND DE- MAILLE, J. G. (1981) B&him Biophyx Actu 673,

303-311. 36. BLUM, J. J., HAYES, A., JAMIESON, G. A., AND VAN-

AMAN, T. C. (1981) Arch. Biochem Biqvhys. 210,

363-371. 37. EICHHORN, J. H., AND PETERKAFSKY, B. (1979) J.

Cell BioL 81, 26-42. 38. FEINSTEIN, M. B., AND PAIMRE, M. (1969) Fed Proc

28, 1643-1648. 39. BROWNING, J. J., AND NELSON, D. L. (1976) Proc

Nat Ad Sei USA 73.452-456. 40. SIDDLE, K., AND HALES, C. N. (1974) Biochxm. J.

142. 345-351. 41. FRIEDMANN, N., AND RASMUSSEN, H. (1970)

B&him Biqphys. Acta 222,41-52. 42. FAIN, J. N., TOLBERT, M. E. M., POINTER, R. H.,

BUTCHER, F. R., AND ARNOLD, A. (1975) Me- taMtim 24, 395-407.

43. CHAZOTTE, B., AND VANDEFXOOI, G. (1981) B&whim Biophys Acta 636,153-161.

44. VACQUIER, V. D., AND BRANDRIFF, B. (1975) Dev. Biol 47, 12-31.

45. SIRACUSA, G., WHITTINGHAY, D. G., CODONESU, M., AND DEFELICI, M. (1978) Dev. Biol 65,531-

535. 46. BABCOCK, D. F., FIRST, N. L., AND LARDY, H. A.

(1975) J. Bid C&m. 250, 6488-6495.

47. SHONK, C. E., AND BOXER, G. E. (1964) Cancer Re.s. 24, 709-721.

48. SCARPA, A., BRINLEY, F. J., AND DIJFJYAK, G. (1978) Biochemistry 17,1378-1386.

49. BERGMEYER, H. U. (1974) Methods of Enzymatic Analysis, pp. 1492-1493, Academic Press, New York.

50. MILKOWSKI, A. L., BABCOCK, D. F., AND LARDY, H. A. (1976) Arch Biochem Biophys. 176,250-

256. 51. SEEMAN, P. (1972) Pharmaod Rev. 24, 583-617. 52. MALAGODI, M. H., AND CHIOU, C. Y. (1974) Eur.

J. Phurmad 27,25-33.

53. CHARO, I. F., FEINMAN, R. D., AND DETWILER, T. C. (1976) Biochen~ Biophys. Res Commun

72, 1462-1467. 54. SKOU, J. C. (1961) J. Pharm Pharr?wcol 13, 204-

217.

55. SKOU, J. C. (1954) Acta Pharmacd Tozied 10, 297-300.

56. GARBERS, D. L., FIRST, N. L., GORMAN, S. K., AND

LARDY, H. A. (1973) Biol Reprd 8,599-606.

57. BRACKETT, B. G., HALL, J. L., AND OH, Y. (1978) Fert. Sted 29,574-582.

58. GARBERS, D. L. (1972) Thesis, pp. 144-158, Uni-

versity of Wisconsin, Madison. 59. NELSON, L. (1972) Exp. CeU Rex 74,269-274. 60. PETERSON, R. N., AND FREUND, M. (1975) BioL Re-

prod 13.552-556. 61. CASCIERI, M., AMANN, R. P., AND HAMMERSTEDT,

R. H. (1976) J. Biol Chem 254,787-793. 62. HOSKINS, D. D., STEPHENS, D. T., AND HALL,

M. L. (1974) J. Reprod Fe& 37, 131-133. 63. CASILLAS, E. R., ELDER, C. M., AND HOSKINS,

D. D. (1980) J. Reprod Fed 59, 297-302. 64. BABCOCK, D. F., SINGH, J. P., AND LARDY, H. A.

(1981) in Calcium Binding Proteins: Structure and Functions (Siegel, F., Carafoli, E., Kret-

singer, R. H., MacLennan, D. H., and Wasser- man, R. H., eds.), pp. 479-481, Eisevier, Am-

sterdam. 65. SINGH, J. P., BABCOCK, D. F., AND LARDY, H. A.

(1980) BioL Reprd 22.566-570. 66. Low, P. S., LLOYD, D. H., STEIN, T. M., AND ROG-

ERS, J. A. (1979) J. Biol. Che-m 254,4119-4125. 67. JOHNSON, C. L., AND SCHWARTZ, A. (1969) J. Phar-

muax! Exp Ther. 167.365-373.

68. MELA, L. (1968) Arch Biochem Biophys. 123,286-

293. 69. KOPF, G. S., AND GARBERS, D. L. (1980) Biol Re-

prod 22, 1118-1126.

70. SCHACKYANN, R. W., EDDY, E. M., AND SHAPIRO,

B. M. (1978) Deu. Bid 65, 483-495.

71. SHELLENBERGER, D. L., AND SHAPIRO, B. M. (1980)

Gamete Res 3.1-7.

72. KOHLHARDT, M., BAUER, B., KRAUSE, H., AND FLECKENSTEIN, A. (1972) Pjugers Arch, Eur. J. Physiol 335,301-322.

73. NACHSHEN, D. A., AND BLAUSTEIN, M. P. (1979)

MoL Pharmnd 16, 579-586.

74. GALPER, J. B., AND CA’ITERALL, W. A. (1979) Mel Pharmaed 15, 174-178.

75. HYNES, R. V., AND GARBERS, D. L. (1979) Proc.

Nat Acad Sci USA 76, 5699-5703.

76. RINK, T. J. (1977) J. Reprod Fed 51, 155-157.

77. VOLPI, M., SHA’AFI, R. I., EPSTEIN, P. M., AN- DRENYAK, D. M., AND FEXNSTEIN, M. B. (1981) Proc Nat. Ad S& USA 73, 795-799.

78. TANAKA, T., AND HIDAW, H. (1981) Bioehem Bti phys Res Commun. 101,447-453.

motility activation, respiratory stimulation, and alteration of ca2+ transport in bovine sperm...

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