BIOLOGY

OF

REPRODUCTION

44,

Hydrogen

Embryology Department

(1991)

Peroxide

BIZE,2’3’4’5

ISABEL

398-403

Is Involved

GABRIEL

in Hamster

SANTANDER,3

PILAR

Laboratory,3 Faculty of Biological of Obstetrics,4 Gynecology and and

Department

Harvard

of Obstetrics

CABELLO,3

Sciences, Reproductive

Medical

and

Sperm

School,

Gynecology,5

Pontflcal Biology,

Catholic Brigham

Science

and

CINDY

SHARPE5

University, Santiago, Chile and Women’s Hospital

Massachussetts

Health

In Vitro1

DRISCOLL,4

DAVID

Boston,

SUM”

Capacitation

02115

Center,

Syracuse,

New

York

13210

ABSTRACT We role

have

investigated

during

culminates do not

Capacitation

in an exocytotic

event,

catalase,

the

AR, while

glucose

also

stimulated

the

by the

appearance

Catalase

added

with

gether,

the

enzyme

catalase

that

prevented

results

suggest

reorganization

that

that

both

the The

effective

induction fusion

that

of the

AR by

the

Capacitation

is a term

that

incubation some

used

occur

during

required

for

reaction

(AR)

companied

by

by

Series

egg-associated

sperm

plays

exocytosis

changes

in

membrane

During

lipid

and

[81. These subcellular patterns and the onset vicinity

of the

egg

successful

sult

the

catalase

Direct

addition

AR was

always

h after

at the

during

mouse,

and

human

hydrogen

sperm

protein

and

(02-)

and

cubation by the

[10-13]. Most of the H2O2 action of sperm superoxide

peroxide

radical

peroxide

produced

(H2O2)

by the

mal conditions, represents only

the H2O2 generated 1-2% of the total

animal

in most

lase

tissues; and

effects

peroxidases of H2O2

Accepted

October

Received ‘Part

[15].

and

motility

to be

the

1):212

and

project

85/83 Research;

Biol

Under

the

well

in excess

concentrations

In this work, enger; H2O2;

the

apparent

lack

appeared

Reprod

1990

the

and

NIH

in (suppl

Pontifical

preliminary 1)88.

Catholic

form Funds

University,

in

were Chile;

Biol

Reprod

partially

toxic

Hendricks

1985 Fund

(suppl

by DIUC for Med-

Bize,

Department

of Biology,

Hamilton

College,

of to,

in mem-

intracellularly;

superoxide,

and

report

Clinton,

398

lipid than

peroxidation

reper-

H2O2, [11-

inhibition of in vigrowing evidence that [181 and in regulating

action of insulin and other us to examine the role of

capacitation. in sperm capacitated in vialbumin [22], This morpho-

effects

(GO), and spontaneous

head at the end of cato study capacitation. of catalase,

an H2O2

scav-

enzyme that generates AR of cauda epididy-

effect of lysophosphatidyl choline in the presence of catalase was

The results allow us to speculate that release of H2O2 is not merely a means

toxic waste products, role during sperm has been recently

increased

sperm-zona

by H2O2

NV 13323.

the

rather

of lipid

the

the

membrane

inducer

discarding a significant hypothesis limited protein

2SO7RR0540227. Isabel

we

glucose oxidase and H202 on the

also examined. generation and

of cat-

provided

11,0,.

Taken

possibly

of the sperm used extensively

mal hamster sperm, The (LPC) in sperm incubated

of cata-

to prevent

the

norcells in

5, 1990.

work

from

‘Correspondence:

[141.

choline.

produced

loss;

main

logical modification pacitation has been

3, 1990.

March of this

sperm

by nonphagocytic oxygen consumption

Nevertheless,

in-

appears to be generated dismutase on the su-

tissues,

are

aerobic

of

Incubation

is spontaneous

metabolite in vitro sperm The AR occurs spontaneously tro in the presence of serum

superoxide

during

of H,O, preceded

addition

capacitation,

H2O2

reactivity

cell division, chemotaxis, hormones [19-211-prompted

interaction

generate

and

inhibited

contents.

this

Rabbit,

the

beginning.

14, 16, 17]. Our preliminary findings-including tro fertilization by catalase and the H202 is involved in metabolic control

[9].

ical

1-2

acrosomal

superoxide of this

appears

changes in must occur

gamete

AR.

of the

Capacitation incubation

added

lysophosphatidyl

role

of the

oxidation

sites is ac-

in ion fluxes, [6, 7], and

changes regulate of the fusion that

for

with

acro-

fuses at multiple [11. Capacitation

of the

added lipid,

aerobic

that

manifest

a significant

during

show

onset

a physiological

alase activity in sperm has made possible the detection of H202 outside the intact rabbit, mouse, and human sperm [10, 12, 13], Mouse, rabbit, and human spermatozoa react

sperm

of the

factors.

membrane membrane

was catalase

AR than

during

of preparatory

triggering

onset The

AR by H,O,

by hamster

of mammalian

and localization [2-41 and changes calcium [5], sodium, and potassium

protons motility in the

the

period

successful

AR, the overlying plasma with the outer acrosomal composition including

for

the

the 11,0,.

we

plays in mammals.

H,O,

work

the

takes

place

present by

fertilization

generate

accelerated and

by spermatozoa

for

membrane-perturbing

INTRODUCTION changes

In the

11,0,,

in inhibiting

(H,O,)

required sperm

by GO

stimulation

produced

the

of H20,.

generates

of the

peroxide

period Mammalian

stimulation

motility. less

(AR).

scavenging

was H,O,

to facilitate

incubation

reaction

an enzyme

the

of hydrogen

as the

promotes

inhibited

of hyperactivated

catalase

generation

acrosome

(GO),

oxidase

AR,

the

is defined

at 3 h of incubation

these

brane

the

that

possibility

capacitation.

contain

sperm

the

interaction

the of

but instead that H2O2 plays capacitation. Support for this provided by work showing following

induction

of

peroxidation in human sperm [23] and activation of kinase C by mild oxidation of its regulatory domain [24].

HYDROGEN

MATERIALS Media

and

STIMUlATES

METHODS

Reagents

Modified

Tyrode’s

vine serum dia contained

albumin 117.5

mM MgCl2, Hepes (pH

11.9 7.4),

9 mM (fraction

AND

PEROXIDE

medium

containing

taurine

and

bo-

(BSA) was used in all experiments. MemM NaCl, 10 mM KCI, 2 mM CaCl2, 0.5

mM NaHCO3, 5 mM glucose,

lactate, 0.5 V, Sigma

0.36 mM NaH2PO4, 0.09 mM sodium

mM taurine and A4503), where

5 or 20 indicated.

10 mM pyruvate,

mg/mi The

BSA pH of

the incubation solution at room temperature was 7,4-7.5 in media with 20 mg/mI of BSA and 7.5-7.6 in media with 5 mg/mi of BSA. Osmolarity was 290-300 mOsm/kg. Thymol-free catalase from from bovine erythrocytes,

niger,

bovine liver, superoxide glucose oxidase from

and

all other reagents were obtained Chemical Company (St. Louis, MO). Sperm the presence of 50 M H202 was performed taining

3 mM

benzoic

acid

dicated,

all enzymes

ginning

of the

incubation.

were

were

recovered

and included

Tyrode’s

was

medium of Ca2”,

was raised an aliquot

was

added

alase

added

so that

to give

was

399

CAPACITATION

the

to 2 mM. of Nat-free

of Ca2 after the Tyrode’s

concentration

in the addition medium K.

Cat-

of sperm suspension (10 pA) were observed microscopy to determine the percentage

by of

added

a final

concentration One hour modified

at 0 and

of 10 mM

3 h.

Observations Samples phase-contract ARs

in at least

tions values

100

were made shown are

strongly

motile

sperm

[27].

Determina-

at 4, 5, 6, 7, and 8 h of incubation. the mean percentages ± standard

The devia-

tion.

dismutase

Asp ergillus

Stattstics

from Sigma capacitation in in media con-

5 mg/mI

SPERM

BSA.

Unless

in the

media

cauda

epididymis

in-

at the

be-

For statistical to arcsin angle between

the

analysis, values. means

all percentages The significance

in Table

1 was

were transformed of the differences

analyzed

using

the

t-test

for paired samples. Figure 1 and all other tables were analyzed using randomized block analysis of variance, within each time period, followed by multiple comparisons with the Student-Newman-Keuls test.

Spermatozoa Sperm

6 mo-old virgin male were minced in 0.5-1 for

15 mm

at room

escape removed.

into

tration 200-Fil

of 1-3 drops

than

sperm

were

of H2O2

scopoletin

peroxidase

by 2.6

not

sperm

Effect to

at 37#{176}C in 100- or that yielded less

included

in this

was

0.25

RESULTS

study.

evolved

by GO

units/mI

assay

was

determined

originally

by De Ia Harpe was 1 p.M, and in a total

of 200

us-

described and Nathan horseradish

by 15 p.g/ml of to that induced

.aM H202.

spontawith

two enzymes that scavenge reactive oxygen species. Catalase (10 g/mi), an enzyme that scavenges H202, inhibited the AR of sperm treated with epinephrine (50 PM). Superoxide dismutase (SOD, 50 g/ml), a superoxide scavenger, no effect. AR [28],

Epinephrine, generates

a commonly H202 during

used aerobic

stimulator incubation;

of

therefore, the inhibition of the AR by catalase could be due to inhibition of epinephrine stimulation rather than inhibition

of the

TABLE

1.

basal

capacitation

rate.

Table

1 also

Catalase

inhibits

the

shows

the

AR.* Acrosome-reacted

Epinephrine

sperm

1% at 5 h)

(tM)

Enzymet

Superoxide (50-100

To investigate whether the observed effects were due to its action on capacitation or directly

of catalase on the AR,

we

and

capacitation

by delaying

dition after preincubating medium. Spermatozoa

spermatozoa were incubated

free

medium

Tyrode’s

stead of 10 mM of the incubation,

on the AR

1 shows the percentage of sperm that lost the acrosomal cap after 5 h of incubation

(10-20

modified

Scavengers

Control

Test

n

Catalase

Synchronization

synchronized

Oxygen

pA in each

was determined using a filter fluoroMicrofluor, Alexandria, VA). The catalase-

decrease in fluorescence induced 1 h of incubation corresponded

of Activated

Table neously

had the

fluorescence

Fluorescence (Dynatech

sensitive GO after

motile

before the pieces of tissue were were diluted to final concen-

by Andreae [251 as modified [261. Scopoletin concentration well. meter

to allow

Activity

amount

the

of 4-

excised epididymides and allowed to stand

million/ml and incubated under mineral oil. Animals

Oxidase

The ing

the

temperature

the medium The spermatozoa

80 million

Glucose

from

hamsters. The ml of medium

K,

as described 8 pA of 50 mM

that

Ca2

K

ad-

in Ca2-free, low K in 200 pA of Ca2contained

0.1

mM

in-

above. At 1 h after start CaCI in K-free modified

p.g/ml) dismutase g/ml)

50

75

±

17

50

75

±

12

9

±

81

±

(% at 7-8

6

7

11b

4

h)

Catalase p.g/ml)

(10-20

*lnhjbjtjon rine. tEnzymes 20

s,bWithin

(p

Values represent were added

mg/mI