Decrease in bicarbonate transport activities during epididymal maturation of porcine sperm

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]280-]287

December 3 0 , 1 9 8 8

DECREASE

IN BICARBONATE

TRANSPORT ACTIVITIES DURING PORCINE SPERM

N a o m i c h i Okamura 1

Institute

, Yutaka Tajima

EPIDIDYMAL MATURATION

OF

and Y o s h i k i S u g i t a

of Basic Medical Sciences, University I b a r a k i 305, J a p a n

o f Tsukuba,

Tsukuba,

Received October 17, 1988

K i n e t i c s t u d i e s o f ~-['4C]HCO~ and ~rSS]SO~- t r a n s p o r t show t h e e x i s t e n c e o f a common t r a n s p o r t s y s t e m f o r t h e two a n i o n s - o n t h e p l a s m a membranes o f p o r c i n e e p i d i d y m a l s p e r m . Km v a l u e f o r b i c a r b o n a t e ( 8 . 3 m M ) i s w i t h i n t h e r a n g e o f physiological concentration of bicarbonate. The a n i o n t r a n s p o r t activity is d e p e n d e n t on t h e t e m p e r a t u r e b u t i n d e p e n d e n t o f c o - e x i s t e n c e o f Na+ . I t s pH d e p e n d e n c y and s e n s i t i v i t y to the several anion transport inhibitors are similar to those observed in erythrocyte. Anion t r a n s p o r t a c t i v i t y d e c r e a s e s d u r i n g sperm m a t u r a t i o n in e p i d i d y m i s . © 1988 Academic

Press,

Inc.

Bicarbonate

h a s b e e n shown t o

stimulate

and m e t a b o l i s m t h r o u g h d i r e c t

and s p e c i f i c

It

bicarbonate

has

signal in

been also on t h e

the

level

found that

initiation of

through adeny]ate Recently, partially

purified

bicarbonate adenylate

and

in

in

is

genital

acts

the

at

genital

as

an

of adenylate

cyc]ase(l).

indispensable

activating

ejaculation fluids

and t h a t

control

sperm

the changes activities

cAMP s y s t e m ( 2 4 ) . Salomon

adenylate

interacts cyc]ase

bicarbonate activate

cyc]ase

Garty

activation

of sperm m o t i l i t y

bicarbonate

m o s t o f mammalian s p e r m m o t i l i t y

have

cyclase

reported from

directly

with allosteric

located

on

fluids

the

must

cytosolic

that

bull site

t h e enzyme. A l t h o u g h t h e k i n e t i c s

sperm,

activates

suggesting

that

o f t h e e n z y m e ( 5 ) . As s p e r m

surface

be t r a n s p o r t e d

bicarbonate

into

plasma

the

cell

in order

to the

1. R e c i p i e n t of a Science Research Grant(61770171) from t h e M i n i s t r y E d u c a t i o n o f J a p a n and t o whom c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

of

1280

transport

membranes,

across

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

of bicarbonate

of

Vol. 157, No. 3, 1988

plasma have

membranes been

plasma

of

erythrocyte,

extensively

bicarbonate In

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

transport

this

study,

membranes

of

renal

studied(6-8),

cortex

and

isolated

little

has

been

urinary known

bladder

about

the

i n t o mammalian s p e r m . we h a v e porcine

investigated sperm

in

the

anion

relation

transport to

those

s y s t e m on t h e maturation

in

epJdidymis.

MATERIALS AND METHODS P r e p a r a t i o n s o f s p e r m - P o r c i n e c a p u t , c o r p u s and c a u d a e p i d i d y m a l s p e r m were collected by f l u s h i n g o u t f r o m d u c t u s e p i d i d y m i d i s w i t h Ca~,Mg 2÷ f r e e p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) c o n t a i n i n g 135mM NaC],5mM KC],7mM Na2HPO 4 and 3mM KH2PO4,pH7.2 and w e r e w a s h e d t h r e e t i m e s w i t h PBS by c e n t r i f u g a t i o n at 1200xg f o r 10min. The s p e r m p e l l e t s w e r e s u s p e n d e d i n 0.29M s u c r o s e , 2 0 m M BEPES p H 7 . 4 ( B u f f e r A) and w a s h e d a g a i n . The w a s h e d s p e r m w e r e s u s p e n d e d i n B u f f e r A. D e t e r m i n a t i o n o f b i c a r b o n a t e and s u l f a t e u p t a k e - As t h e i n t r a c e l l u l a r water space of sperm is extremely small(about 2pl/ I0 B c e l l s ) ( 9 ) , it is important for the accurate estimation of radiolabeled anion uptake to eliminate the contamination of extracellular medium. We t e s t e d s e v e r a l m e t h o d s t o s e p a r a t e s p e r m f r o m medium u s i n g [ 3 H ] m a n n i t o ] a s e x t r a c e l l u l a r space marker a c c o r d i n g t o F o r d and f l a r r i s o n ( 9 ) . Among s i l i c o n oil, n-butyl phtalate and various concentrations of sucrose(0.25-1.0M), the rapid sedimentation of sperm t h r o u g h 0.75M s u c r o s e was m o s t e f f e c t i v e t o remove medium. The m e t h o d o f t h e filtration through glass fiber filter was s l i g h t l y more e f f e c t i v e to exclude [3H]mannitol contamination, but data were not reproducible. So, we e m p l o y e d t h e m e t h o d o f r a p i d s e d i m e n t a t i o n t h r o u g h 0.75M s u c r o s e a s f o l l o w s . Sperm w e r e incubated in Buffer A containing various concentrations o f O4C]NaHCO3 o r ~5S] N~SO4(specific activity;1Ci/mole) a t 37°C. At a p p r o p r i a t e t i m e , 50pl a l i q u o t s o f a s s a y m i x t u r e w e r e l a y e r e d on t h e t o p o f t h e d i s c o n t i n u o u s s u c r o s e g r a d i e n t f o r m e d by 20p] o f 2.7M s u c r o s e and 400)11 o f 0.75M s u c r o s e and w e r e i m m e d i a t e l y centrifuged a t 9000xg f o r l m i n . Sperm w e r e p r e c i p i t a t e d on t h e 2.7M s u c r o s e l a y e r . The t i p o f t h e c e n t r i f u g e t u b e was c u t by s c i s s o r s i n t o lml o f 1N NaOH. Sperm p e l l e t s w e r e h o m o g e n i z e d by s o n i c a t i o n and s o l u b i l i z e d by b o i l i n g at 100°C f o r 2mJn. A f t e r n e u t r a l i z a t i o n w i t h HCI, 800p] a l i q u o t s w e r e s u b m i t t e d to counting for radioactivity by s c i n t i l l a t i o n spectroscopy. The a n i o n transport activity was e x p r e s s e d by t h e d i f f e r e n c e between the radioactivity incorporated into sperm pe]let(108cel]s) a t a p p r o p r i a t e t i m e and t h a t a t 0min. materials 4-Acetamido-4'-isothiocyanatostilbene-2,2'disu]fonic acid (SITS), probenecid, acetazolamide, ouabain, furosemide were purchased from Sigma. 4 , 4 ' - d i i s o t h i o c y a n a t o - s t i ] b e n e - 2 , 2 ' - d i s u l f o n i c a c i d ( D I D S ) was o b t a i n e d f r o m D o j i n C h e m i c a l Co.(Kumamoto, J a p a n ) . NAP t a u r i n e was o b t a i n e d f r o m P i e r c e C h e m i c a l Co. [35S]Na 2 SO4 and [3 H ] m a n n i t o l w e r e p u r c h a s e d f r o m New E n g l a n d N u c l e a r : ~4C]NaHCO3 was o b t a i n e d f r o m Amersham.

RESULTS The

incorporation

dependent. 4°C,

it

of

anions

As shown i n F i g . l ,

was

incorporated

into

the

sperm

was

time

while the uptake of bicarbonate

rapidly

and r e a c h e d

1281

a maximal

level

and

temperature

did not occur at within

5min a t

VoI. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,

|

L.I

0,5 \ C) E 0,4

t ~.

0,3

~:.,

0,2

/~~C~

20ramNaHI~C ' 03 ~ ~ ) ~

"

,

20rnMKH~C ' 03 N

o

0,1

I m

212

0

5 Incubation time

10 { rain. )

Fig. l Time c o u r s e o f a n i o n t r a n s p o r t . Sperm(lO 8 c e l l s ) were i n c u b a t e d in 500p] o f lOmM ~SS]Na2SOg(A), 20mM ~gC]NaHC03(© ) o r 20mM ~gC]KHC03($ ) in B u f f e r A a t 3 7 ° C ( s o l i d l i n e ) o r 4 ~ C ( b r o k e n l i n e ) . At a p p r o p r i a t e t i m e , sperm were s e p a r a t e d from i n c u b a t i o n medium and r a d i o a c t i v i t i e s t r a n s p o r t e d i n t o sperm were d e t e r m i n e d a s d e s c r i b e d i n MATERIALS AND METHODS. Each p o i n t i s mean S.E. from 6 d e t e r m i n a t i o n s .

37°C. min,

Sulfate which

transport presence

was t r a n s p o r t e d was,

of

bicarbonate

o f N~

on t h e p r e s e n c e In o r d e r the

initial

function linear

although,

of

than

s l o w l y and r e a c h e d a maximal l e v e l

slightly as

well

higher as

K+ a s a c o u n t e r

than

that

sulfate(not cation

of

shown)

b u t was n o t

at

bicarbonate. was

higher

absolutely

in

20 The the

dependent

o f Na +.

to

show t h e p r e s e n c e

rates anion

between them.

by

sulfate

competitively

inhibited

and the

of sulfate

and Km v a l u e s

vice

specific

and s u l f a t e

concentration(Fig.2).

competitively

ve]ocities(Vmax)

of the

of both bicarbonate

relationship

108cells/min,

rather

transport

The d o u b l e

And t h e versa.

transport

transporter(s)

Addition

transport of

of both anions(not

and b i c a r b o n a t e

transport

were analyzed as a

reciprocal

bicarbonate

for anions,

plot

showed a

was i n h i b i t e d

5mM c l o r J d e shown).

ion

The maximal

w e r e 133 and 7 4 . 1 p m o ] e /

f o r t h o s e w e r e 1 3 . 7 and 8.33mM, r e s p e c t i v e l y .

1282

also

Vol. 157, No. 3, 1988 i

a)

60'

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS !

K~ = 8 , 3 3 m M

~

0

90 " K m

~

Vmax= ?k,1 prnole/ 108celt /rain

_

5O

- -

/$./

/

6o

i.//°/

~ ~s

/

+20raM NaHC0 3

./ + 20~M ~s%

A/

~2o ¢

75

i

./

/

-' 40

/

= 13,7mH

Vmax= 133pmole/ 108¢el1 / rain.

O

/i

i

b)

, ,// ? / 10 / !

~. °"°I

,/ ,"~

~ oo,l

_-

-0,1 I

0

0,1

mH

10 [oncenhafion of NaHC03

/

0,5

/

~.-Y-~, ..... 0 0<1

/

0,1

i

&--" ~

I

~-. & J

/. iA''/

/

' I

..@

./"/'

~g 30

20 ( mM)

I

10

Concen'l'ration of Na2SO~ .

0,5

raM-

20 ( mH )

Fig.2 K i n e t i c parameters of anion t r a n s p o r t . S p e r m ( 1 0 8 c e ] l s ) were i n c u b a t e d e i t h e r w i t h v a r i o u s c o n c e n t r a t i o n s o f O4C]NaHCO3(a) i n t h e p r e s e n c e ( O ) o r t h e absence(O) o f 20mM N~ S0h or w i t h v a r i o u s c o n c e n t r a t i o n s o f ~5S]N~ SO~(b) in t h e p r e s e n c e ( A ) or t h e a b s e n c e ( A ) o f 2 0 4 NaHCO3 a t 37°C f o r 0 or 10min. Radioactivities transported i n t o sperm were d e t e r m i n e d a s d e s c r i b e d i n MATERIALS AND METHODS. Double r e c i p r o c a l p l o t i s shown in t h e lower i n s e t . R e p r e s e n t a t i v e d a t a from 4 e x p e r i m e n t s a r e shown and e a c h p o i n t i s a mean from duplicate determinations.

The

anion

transport

activity

incorporation

of bicarbonate

pH i n c r e a s e d

and

higher

3,

characterizations

sensitivity disulfonic

taurine hand,

the

as well

dependent

as sulfate

maximum a t

pH6.5

on

the

external

increased

as the

and

steeply

then

pH.

The

extracellular decreased

at

pH(not shown),

Further its

reached

is

to the stilbenes

reduced the

the

transport

several

of

sperm anion

inhibitors

of

both

were done to

of anion transport.

s u c h a s DIDS a n d SITS,

transport

transport

frosemide,

bicarbonate

of both anions was insensitive

ouabaine.

1283

and to

As s h o w n i n F i g .

probenecid

sulfate,

either

evaluate

a n d NAP-

On t h e

acetazolamide

other nor

Vol, 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100

.....] ....i

50

IIIIIII Fig.3 Effects of anion transport inhibitors on b i c a r b o n a t e and s u l f a t e t - ~ a n s p o r t i n t o sperm. Sperm(10 ° c e l l s ) w e re i n c u b a t e d w i t h lOmM [%C]NaHCO3 ( op e n b a r ) o r [35S]NaTSO4 ( c l o s e d b a r ) i n t h e p r e s e n c e o f lmM o f i n h i b i t o r s at 37°C f o r 0 o r 5min. R a d i o a c t i v i t i e s t r a n s p o r t e d i n t o s pe rm w e r e d e t e r m i n e d a s described i n MATERIALS AND METHODS. D a t a a r e e x p r e s s e d by p e r c e n t a g e o f transport activity in the absence of inhibitors and a r e mean ± S.E. from 4 determinations.

cAMP has

been

suggested

to

concern

with

the

sperm

maturation

in

epididymis as an i n t r a c e l l u ] a r r e g u l a t o r ( l O ) . As bicarbonate r e g u l a t e s cAMP l e v e l s in sperm through a c t i v a t i o n of adenylate c y c l a s e , t h e anion t r a n s p o r t a c t i v i t i e s in sperm obtained from t h e d i f f e r e n t regions of compared.

As

transported corpus

and

shown

in

Fig.4,

the

immature over

both

bicarbonate

and

sulfate

cauda

epJdidymJs.

The

activity

same as that

in

cauda

epididymis(data

are

in not

sperm

three the

in

times

freshly

epididymis were

caput as

epididymJs

high

as

ejaculated

those

in

sperm was

shown).

DISCUSSION This

is the

first

transport

system

is confirmed

was

incorporated

process

is

into

seemed

to

communication

sperm be

that

in mammalian

in time mediated

the

existence

sperm.

Radio-labeled

and temperature by

a

carrier

of the bicarbonate

dependent protein,

bicarbonate manner.

because

This doub]e

reciprocal plot of initial rate of transport against bicarbonate concentration

1284

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

~1} Caput epidi~ymat

b) Corpus epididymatsperm

sperm

4

C} [auda eoididymalsperm

.¢-

*'3 "6 t=

/

v

t]3

b

lb

0

%

2'o

10 ' Incubation time

lb

2.0

2'o

( min. )

Fig.4 Comparison of b i c a r b o n a t e and s u l f a t e t r a n s p o r t a c t i v i t y among c a p u t , c o r p u s and cauda e p i d i d y m a l sperm. 1 0 8 c e ] ] s o f sperm from c a p u t ( a ) , c o r p u s ( b ) or c a u d a ( c ) e p i d i d y m i s were i n c u b a t e d w i t h lOmM ~4C]NaHCO3 (A) or ~5S]Na2S ~ (O) a t 37°C. At t h e t i m e i n d i c a t e d , r a d i o a c t i v i t i e s t r a n s p o r t e d i n t o sperm were d e t e r m i n e d . Each p o i n t i s mean ± S.E. from 3 d i f f e r e n t e x p e r i m e n t s each w i t h triplicate determinations.

shows a straight with

sulfate

and

concentration

Several

been

in

basic

in

that

co-existence

participate (8),

genital

of

in

because

for

fluids,

of

those

basolateral

Bicarbonate

than

Km v a l u e

properties

comparison

cortex(7).

the

of

bicarbonate

shares

the

common t r a n s p o r t e r

bicarbonate(8.33M) which

was

suggests

that

transport

in

within

this

its

transport

works in vivo.

reported

higher

Furthermore,

cloride.

range

system actually

studied

line.

in

into

presence

tissues. vesicles

but

carbonic

sperm bicarbonate

it

were

also

HC0~ c o - t r a n s p o r t

has

isolated

is

not

did

not

rabbit

o f Na + i s

absolutely

show

renal

slightly

dependent

nor N~-K+-ATPase

differently

ouabain

sperm

from

presence

anhydrase

transport,

and

Na + /

sperm in the

o f K÷,

o f Na + . N e i t h e r

acetazolamide

other

membrane

transport

in the

bicarbonate

on

may n o t

from urinary

bladder

any

on

effects

the

transport. The

inhibitors

of

anion

exchange

stilbenes,

frosemide,

probenecid

bicarbonate

transport.

The e x t e r n a l

in

sperm

was

characterization sperm transporter

same

as

that

and

in

NAP-taurine

and erythrocyte

all

these

in

data

suggest

as

inhibited

disulfonic the

bicarbonate

erythrocyte(6).

one(Band3 protein).

1285

such

all

pH d e p e n d e n c y o f t h e

reported

m u s t be d o n e ,

erythrocyte

the

Although similarity

sperm

transport further between

Vol. 157, No. 3, 1988

The

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

importance

glycolytic

of

bicarbonate

activity,

capacitation

has

been

well

that

molecule

reactions

the

these

activation

porcine

of

is

bicarbonate

in

concentrations

of

independently bicarbonate passage

through

transport

and

in decrease to

fluid

to

and

of

Although, remained

likely

Along with

In

is

of

be

change

during

the anion transporter these

lines,

yet,

identification

and

to

as

shown

in

be

caput

this

of

the

readsorbed

sperm which have intracellular

matured

both

cAMP

At

and

the

have

the

initiation

of tracts

of sperm occur

are

40mM)(13).

transport suggested

during

transporter

in

sperm

activity that

sperm

epididymis(14-22).

somehow m o d i f i e d

of sperm anion

into

reproductive

of bicarbonate(over

are

in the

as high as that

metabolism

many e v i d e n c e s

sperm

ejaculation,

incorporation

female

testis

bicarbonate

sperm. 10 t i m e s

the

epididymis,

from 20mM i n f e t e

of

in

study, with

is partially

epididymis,

that

The

regulated

significantly

in the bicarbonate

1286

activities.

known

and zona penetration

also

seem t o b e v e r y

are

increase

upto

extracellular

bicarbonate

maturation is

both

of

high concentration

elucidated

So,

the

interesting

the mechanism of decrease to

in

in

low l e v e l s

sperm

acrosome reaction

maturation

into

cauda

which facilitates

It

functions,

incorporated

increases

of

in

decrease

activity

acutely

activation

further

properties that

because

observations

of bicarbonate

fluid.

transport

movement(a).

known t o c o n t a i n

in

through

our

from testis

also

concentration

most probably

with

And,

Namely,

is

acts

fluids

sperm

results

epididymal

fluid,

where capacitation,

surface

This

be q u i e s c e n t

the

progressive

has

the

and

it

maturation

which originates

bicarbonate

induces

of

and

a pH-buffering

of sperm surface

blood(4,12,]3).

reaction

a s more t h a n j u s t

by cAMP(a0).

genital

respiration,

and ¥anagimachi

sperm

sperm

activities

activity.

cauda epididyma] and

in

bicarbonate

extrace]lular

of

epididymis(Fig.5).

2-6mM i n

maintained

these

as

acrosome

accordance

activity

in

epithelium(~2)

high

to

transport

bicarbonate

by e p i d i d y m a l

fluid

that

in

such

Bhattacharyya

that

be regulated

regulation

transport

extracel]ular

cyclase

bicarbonate

of

functions

addition

and its

the

motility,

and suggested

known t o

functions

Recently,

bicarbonate

In

also

level

important

noticed.

adenylate

sperm(I,2).

epididymis

sperm

cAMP metabolism,

(11) have reported in

in

this

It

is

process.

protein

and

Vol. 157, No. 3, 1988

analysis

of the d e t a i l e d

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mechanism how t h e t r a n s p o r t e r

is modified during the

p r o c e s s o f sperm m a t u r a t i o n i n e p i d i d y m i s a r e i n p r o g r e s s now.

REFERENCES

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