An acrosin-acrosin inhibitor complex in ejaculated boar sperm

Vol. 45, No. 2, 1971

BIOCHEMICAL

L. J. D. Zaneveld,*

K. L. Polakoski, Department

August

RESEARCH COMMUNICATIONS

INHIBITOR COMPLEX IN EJACULATED BOAR SPERM

AN ACROSIN-ACROSIN

Received

AND BIOPHYSICAL

and W. L. Williams

of Biochemistry, University Athens, Georgia 30601

of Georgia

10, 1971

Summary: Acrosin (a proteolytic enzyme) activity is high in extracts of epidic&naZ sperm acrosomes but low in extracts of ejacuZated sperm acrosomes. This lower activity from ejaculated sperm has previously been postulated as resulting from the reaction of a seminal plasma proteolytic enzyme inhibitor with acrosin in the acrosome. Evidence is presented which demonstrates that an acrosin-acrosin inhibitor complex exists in extracts of ejaculated boar sperm acrosomes. Upon incubation at pH 3 the complex dissociates and the free enzyme is demonstrable with the use of the synthetic substrate, benzyl arginine ethyl ester (BAEE). The increase in activity from incubation at pH 3 does not result from zymogen activation. Epididymal sperm have the ability to pick up the protease inhibitor from solution. The fact that there is no excess of free acrosin nor of free inhibitor in extracts of ejaculated sperm acrosomes is further evidence for the existance of an acrosin-acrosin inhibitor complex. Introduction:

Acrosin

common with or natural

both

lucida is

(4)

of the

present

plasmin

protease

and in vitro

is

a unique and trypsin.

inhibitors

A previous

rabbit

ejaculated

sperm.

The high

incubation

of intact

that

a seminal

epididymal

sperm

plasma

activity

in seminal

acrosin

(6)

inhibitor

with

has shown

that

sperm,

(7).

was added

in

synthetic

(2)

in uivo

through

of epididymal

plasma

properties

both

of sperm

and capacitated

acrosin

with

fertilization

passage

report

(1)

of acrosin

prevents

by blocking

in both

protease

Inhibition

(3,4)

presumably

ovum (5).

acrosomal

(2,3)

the zona acrosin

but

is

pel-

activity very

low in

sperm was lost

Zaneveld

et aZ.

to acrosin

during

(7)

upon

proposed

ejacula-

tion. Kunitz complexes ments extracts

that

and Northrop dissociate

(8)

under

show the existance

demonstrated acidic

that

conditions.

some protease-protease This

of an acrosin-acrosin

report inhibitor

describes complex

inhibitor experiin

of acrosomes.

*Department Chicago, Illinois

of Ob & Gyn, University 60637.

of Chicago,

381

Chicago

Lying-in

Hospital,

Vol. 45, No. 2, 1971

Methods:

Sperm obtained

removed

by the method

Zaneveld, from

the epididymides

acrosomes

removed

0.0017

change

small

of enzyme

and rabbit

to pH 3.0 with adding

of optical solution

0.1

by Polakoski, flushed

(10).

M borate

Acidic

acrosomes

sperm were

by detergent

in 0.05

(11).

and the

as modified

and 0.1 ml added

M BAEE (Mann)

of 5 min and then the

suspended

centrifuged

was done by adjusting

inactive

(9)

and the

and observing

buffer

Acrosomal at pH 7.6

to 2.9 ml 0.05 incubation

1 M HCl,

M Tris-HCl

of the

incubating

at this

ml to the BAEE solution

density

at 253 mu (10).

to the substrate

prevents

above pH

at pH 8.0

Addition

of this

formation

of the

complex.

Results

and Discussion:

extracts

of epididymal

Curve

and Srivastava

were

RESEARCH COMMUNICATIONS

were washed

boar

containing

aliquot

ejaculates

Epididymal

M CaCl,

a minimum

AND BIOPHYSICAL

(10).

0.05

extracts

boar

of Hartree

(1 mg protein/ml)

containing buffer

from

and Williams

extracts

for

BIOCHEMICAL

B in Fig.

1

Curve A in Fig. boar

shows

sperm

1

acrosomes

the acrosin

shows

the acrosin

after

preincubation

activity

in extracts

activity

in

at pH 7.6.

of epididymal

boar

(B)

0.40-

(A)

0.00

0

1

2

3

4

5

6

7

8

9

10

Minutes

Fig.

1.

Acrosin activity of an epididymal succession as follows: A (0) B (0)

1st Incubation, 2nd Incubation,

pH 7.6, pH 3.0,

assay assay

382

boar

acrosomal

at pH 8.0 at pH 8.0

extract

incubated

in

Vol. 45, No. 2, 197 1

sperm

acrosomes

prior

to the

extracts

after

the

assay.

for

the

dissociation

possibility examine bation

boar increase

at pH 3.0 is

this

that

indicating

that

complex

was formed.

the extract

either

denatured.

acrosin

inhibitor

sperm

after

Curve

complex

for

results

activity

strongly

in extracts

result

suggest

the

of ejaculated

incu-

activity. resulted,

or that

an inhibitor

presence

,)J

after

activity

that

sperm

2

To

acrosin

of again

indicating

Fig.

A second

extract

of the enzyme occurred

acrosin

B in

of acrosin.

no acrosin

2 shows the

in One

complex.

acrosomal

2, essentially

D of Fig.

the high

in Curve

at pH 7.6 and tested

C of Fig.

activity

treatment.

inhibitor

a zymogen form

and preincubated

acrosin

similar shown

the ejaculated

incubated

These

show the

activity

pH activated

denaturation

at pH 3.0,

was not

2

acrosomes

RESEARCH COMMUNICATIONS

to pH 3.0

A and B in Fig.

possibility

in Curve

adjusted

of an acrosin-acrosin

at pH 3 was again

As can be seen

were

in acrosin

acidic

second

AND BIOPHYSICAL

extracts

Curves

of ejaculated

explanation is

BIOCHEMICAL

incubating the

enzyme

of an acrosin-

acrosomes

and very

(B)

Minutes

Fig..

2.

Acrosin activity of an ejaculated succession as follows: A B C D

(0)

1st 2nd t ) 3rd (a) 4th 0)

Incubation, Incubation, Incubation, Incubation,

pH pH pH pH

7.6, 3.0, 7.6, 3.0,

boar assay assay assay assay

383

at at at at

acrosomal pH pH pH pH

8.0 8.0 8.0 8.0

extract

incubated

in

Vol.45,No.

likely

2, 1971

in the acrosome.

will

be published Since

results

a brief

available

means were inhibitor.

the

acrosomes

the

acrosin

(B)

as described

identical is

between

incubation

of the sperm

acrosin

were

removed

activity

to line taken

for

previous

B of Fig.

that

adsorbed

Lines

Ringer

the

purified

for

15 min with

Phosphate

it

and

3 show

at pH 8 (A) and pH 3

The control results

we

(12),

A and B of Fig.

preincubation

sperm

and since

plasma

was incubated

(10).

From these

plasma

seminal

in 50 ml of Krebs

after

rabbit

exists

0.00

inhibitor

and ejaculated

seminal

sperm

inhibitor

experiments. 3.

in the

epididymal

sperm

of the extracts

sperm

from boar

and extracted

up by epididymal

The possibility

if acrosin

rabbit

epididymal

inhibitor

to determine

epididymal

of the

(11).

Six mg of purified

2 x 10s washed

and characterization

difference

a purified available

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The purification

elsewhere

the major

from

have

tor

BIOCHEMICAL

without appears

inhibitor the boar

was inhibi-

sperm.

the inhibitor

is

bound

non-specifically

to

(A)

0

12

3

4

5

6

7

8

9

10

Minutes

Fig.

3.

Acrosin activity of an extract of epididymal rabbit sperm that previously incubated with purified boar seminal plasma acrosin inhibitor. A (0) B (0) C (A)

1st Incubation, pH 8.0, assay at pH 8.0 2nd Incubation was done at 3.0, assay at pH 8.0 Same as B but in presence of 2.5 ug purified boar acrosin inhibitor 384

seminal

were

plasma

Vol.45,No.2,

1971

the exterior excess

BIOCHEMICAL

of the acrosome.

of enzyme or of

The insignificant pH 7.6 suggest excess

there

The hyperbolic

curve

inhibitor

competes

obtained

if

the

and the that with

extract

at pH 3.0

tor

suggest

there

is no excess study

(line

elsewhere

acrosin-acrosin

inhibitor

complex

reported

herein

inhibitor

from

unique

for

seminal

plasma

is

(7)

added

after

to acrosin

mixture of

added

of sperm from the occurrence

sperm

support

are

incubation

of ejaculated

of boar

et ~2.

results

of

inhibi-

extracts.

appears

in acrosomes

extracts

and by Zaneveld

It

to

the excess

or incubation

on extracts

(13).

of an

at pH 3.0.

1,2 and 3) without in these

at

was added

Similar

obtained

B in Fig.

and discussion

be reported

and not

line

acrosomes.

effect

3) indicates

substrate

of inhibitor

will

phenomenon

to either

the

preincubation

the enzyme.

an

incubation

inhibitor

after

either

sperm

2) after

trypsin

C of Fig.

The straight

initial

Fig.

out

for

expect

To determine

carried

added

extracts.

one would

A in

boar

(line

RESEARCH COMMUNICATIONS

of ejaculated

of enzyme.

substrate

the

species

(line

results the

true,

the extracts

assay

is

A more extensive

were

pg of purified

inhibitor

of the ejaculated

in

of activity

2.5

mixture

this

is no excess

of inhibitor,

the substrate

If

inhibitor

amount

AND BIOPHYSICAL

the

of an

sperm

acrosomes.

is

a general

The results

hypothesis

at the time

11

that

acrosin

of ejaculation

(14). The high acrosomes

(6)

capacitated removed

acrosin

activity

and the

fact

sperm in during

capacitation

The physiological itor after general

is

presently

a better is

viva

only

at alkaline that

trypsin

(2,3)

and in

in the

inhibitors vitro

female

advantage

of this

a subject

for

understanding

of the

pH in extracts

(4)

prevent indicates

of capacitated

sperm

fertilization

by

the inhibitor

is

tract. apparent

speculation biochemical

regulatory

role

of the inhib-

and may be clarified processes

of fertilization

only in

achieved.

Thanks are due to Dr. C. W. Foley, Acknowledgements: School of Veterinary Medicine, University of Georgia, boar sperm, and to Dr. P. N. Srivastava, Department of of Georgia, and Dr. G. F. B. Schumacher, Department of ogy, University of Chicago, for their advice concerning involving the preincubation of sperm acrosomal extracts 385

Department of Physiology, for the collection of Biochemistry, University Obstetrics and Gynecolthe experiments at pH 3.0.

Vol. 45, No. 2, 1971

This research was National Institute of Award No. 2-K3-GM4831 Contracts NIH-70-2147 Department of Health, 0805A.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

supported by Training Grant No. 5-TOl-HD00140 from the Child Health and Human Development; Career Development (to WLW) from the National Institute of Medical Sciences; and NIH-69-2103 with the National Institutes of Health, Education and Welfare; and Ford Foundation Grant No. 680-

References 1.

2. 3. 4. 5. 6. 7. i: 10. 11. 12. 13. 14.

Zaneveld, in press Zaneveld,

L. J. D., Polakoski, K. L., and Williams, W. L., Biol. Reprod. (1971). L. J. D., Robertson, R. T., and Williams, W. L., FEBS Letters, 12, 345 (1970). Zaneveld, L. J. D., Robertson, R. T., Kessler, M., and Williams, W. L., J. Reprod. Fert., 25, 387 (1971). Stambaugh, R., Brackett, B. G., and Mastroianni, L., Biol. Reprod., 1, 223 (1969). Gould, K., in manuscript. Zaneveld, L. J. D., Srivastava, P. N., and Williams, W. L., J. Reprod. Fert., 20, 337 (1969). Zaneveld, L. J. D., Srivastava, P. N., and Williams, W. L., Proc. Sot. Exp. Biol. Med., 133, 1172 (1970). Kunitz, M., and Northrop, J. H., J. Gen. Physiol., 19, 991 (1936). P. N., J. Reprod. Fert., 9, 47 (1965). Hartree, E. F., and Srivastava, Polakoski, K. L., Zaneveld, L. J. D., and Williams, W. L., Biol. Reprod., in press (1971). Polakoski, K. L., and Williams, W. L., in manuscript. Polakoski, K. L., Zaneveld, L. J. D., Williams, W. L., and McRorie, R. A., Fed. Proc., 30, 1077 (1971). Zaneveld, L. J. D., Polakoski, K. L., and Williams, W. L., in manuscript. Williams, W. L., Biochemistry of Capacitation of Spermatozoa, Proceedings of the Harold C. Mack Symposium on the Physiology and Pathology of Reproduction, Wayne State University. Academic Press (1971).

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