BIOLOGY

OF

REPRODUCTION

30,

833-841

(1984)

Lipid

Peroxidation Hydrogen
JUAN G.

and the Reactions of Superoxide Peroxide in Mouse Spermatozoa

ALVAREZ and BAYARD T. STOREY

and

Departments

of Obstetrics
University

and
of

Gynecology,
Pennsylvania

and

Physiology

School
Philadelphia,

of Medicine
Pennsylvania 19104

ABSTRACT
Mouse spermatozoa released from the cauda epididymidis underwent spontaneous lipid peroxidation during aerobic incubation at 37#{176}C in medium containing 113 mM NaCI, 0.4 mM EDTA, and 15 mM sodium phosphate (NTFC). The rate of lipid peroxidation, as measured by malonaldehyde production, was 0.045 nmol malonaldehyde/h per io cells. The motility of these cells declined with time in medium NTPC; the percent spermatozoa showing no motility increased linearly with production of malonaldehyde. All flagellar activity stopped at 0.80 nmol malonaldehyde/1o8 cells, independent of the malonaldehyde production rate. Spermatozoa suspended in NIPC at 24#{176}C produced O2, with an intrinsic rate of 1.96 nmol/min per io cells; this increased to 3.80 nmol/ mm per i0 cells in 10 mM cyanide. Mouse sperm contain 3.5 U/108 cells of superoxide dismutase activity, 91% of which is sensitive, and 9% of which is insensitive, to cyanide inhibition. Mouse sperm also produce lI2 02, all of which can be attributed to the action of superoxide dismutase on 0 produced. Mouse sperm contain high levels of glutathione and of glutathione reductase and peroxidase activities, implicating the glutathione system as the major protective enzyme system against cell damage by autoxidation. This is in contrast to rabbit spermatozoa, which have little endogenous glutathione and rely on superoxide dismutase as protective enzyme against peroxidative damage.

INTRODUCTION
While peroxidation been of Jones Mennella Mann, quite species et studies in extensive (Jones and 1978, Jones, studies of mammalian and have promoter-induced spermatozoa covered 1973, Mann Mann of et and

rabbit lipid
have a number 1976, 1977; 1983a),

spermatozoa
which have

(Storey
no detectable

and (Holland
low

Alvarez,
catalase

activity
Storey, endogenous g!utathione tase 1981). peculiar activities The to other

and
1981).

produce
They glutathione, peroxidase (Li, 1975;

U2O2
have although and

and
possess reduc-

detectable they

Mann, 1979, 1980;

al.,
and 1981),

a!.,

1980;
lipid

glutathione Holland and

Lutwak-

Storey,

spontaneous

low
the

endogenous
and the

glutathione
boar. Spermatozoa

seems to order have of

peroxidation
primarily to

have rabbit 1982,

that

thus
spermatozoa

far We

been have
(SO)

confined
and

rabbit

(Alvarez

from

species
nmol/108

have contents

been

reported of the the for

have

Storey,
eivdence intracellu!ar!y peroxidation the more anion oxide matic

1983a,b).
superoxide

presented
produced

total
0.5-1.3

glutathione has
1975).

is the
(Alvarez form than O2

principal
and Storey, HO2 its

inducer
1983a), being

of

lipid
with

rabbit
(Li,

been
Rabbit

cells while given as 0.1 and

spermatozoa

figure for boar as 0.3

also been

perhydroxyl effective form

300-fold

conjugate et al., the 1983). primary peroxidation

base,

the
Superenzyin

reported exogenous were a!.,

o!ic (Carey these

to

be relatively resistant 1-12 02, while mouse to Since be we relatively have

of

to damage spermatozoa (Wales metab1981),

spermatozoa Storey,

by
Ct

(Alvarez (SOD) against

found
1959).

sensitive some
mouse

dismutase
defense

is
lipid

previous

characterization et cells

al.,

1981;
to

Heffner
offer

and
a useful

seemed

comparison

to
Accepted Received Reprint Obstetrics University Philadelphia. January 16, 1984. December 16, 1983. requests: Dr. Bayard T. Storey, and Gynecology, Medical Labs of Pennsylvania, School of PA 19104. ous Dept. of 3391G3, Medicine,

rabbit
lipid

spermatozoa
peroxidation,

with
loss

regard
of

to spontaneproducof the protect 1979):

motility, activities to et a!.,

tion of enzymes against

SOD,

SO 02

and which

H2 02, are
and

and the considered (Chance

glutathione

toxicity

catalase,

peroxidase/

833

834

ALVAREZ

AND

STOREY

reductase. an in investigation epididyma!

In

this of mouse

paper, these

we

report of

the 02

results reactions

of

aspects

spermatozoa.

MATERIALS Media

AND

METHODS

Four different media were used for sperm suspensions in this study. Once was a modification of NTP, the high Na medium of Alvarez and Storey (1982), which was designated NTPC, with the composition: 113 mMNaCI, 12.5mM NaH2PO4, 2.5mM Na2 HPO4, 1.7 mM CaCI2, 20 mM Tris, 1.5 mM D-glucose, 0.4 mM EDTA, 0.6% penicillin-streptomycin adjusted with HCI to pH 7.4 0.05. For studies in which the concentrations of K+ and Na+ were changed in NTPC, the KCI concentration was varied from 0-100 mM so that (NaCl) + (KCI)=130 mM. The second medium was a complete culture medium (CM) for in vitro fertilization (Wolf and lnoue, 1976), which is a modified Krebs-Ringer bicarbonate buffer containing bovine serum albumin (BSA), glucose, pyruvate and lactate (Toyoda et a!., 1971). The third medium used was a Tris buffer (TNC; Heffner and Storey, 1981), with the composition: 126 mM NaCl, 20 mM Tris, 1.7 mM CaCl2, adjusted with HCI to pH 7.4. The fourth medium was a modification of the latter, designated TNCPE, with the composition: 20 mM Tris, 126 mM NaCl, 1.7 mM CaCl2, 12.5 mM NaH3PO4, 2.5 mM Na2 HPO4, 0.4 mM EDTA, 0.6% penicillin-streptomycin, adjusted with HCI to pH 7.4-7.5. Where designated, BSA at 10 mg/mi was added to NTPC (NTPCA) and TNC (TNCA). Reagents Xanthine oxidase, xanthine, superoxide dismutase (Type I) from bovine erthrocytes, cytochrome c (Type VI) from horse heart, oligomycin, rotenone, catalase (Type C40) from bovine liver, glutathione reductase (Type IV) from yeast, oxidized and reduced glutathione, aminotriazole, sodium pyruvate, sodium lactate, BSA, thiobarbituric acid (TBA), adenine nucleotides and N,N-ethylenediamino tetraacetic acid, disodium salt (EDTA) were from Sigma Chemical Co. (St. Louis, MO). Glutathione peroxidase was from Boehringer Mannheim (Indianapolis, IN). Yeast cytochrome c peroxidase was the gift of Professor C. P. Lee, Wayne State University. Malonaldehyde-bis (dimethylacetal) was from Aldrich Chemical Co. (Milwaukee, WI). Trichloroacetic acid and inorganic salts were from J. T. Baker (Phillisburg, NJ). Filipin was obtained from Polysciences, Inc. (Warrington, PA). Penicillin-streptomycin was from Gibco Labs. (Grand Island, NY). Preparation of Spermatozoa

was 0.6-3 X i0 cells/mi. For experiments in which sperm were to be incubated in media of different ionic compositions, the sperm were recovered by centrifugation at 300 X g for 10 mm and washed once by a duplicate centrifugation and resuspension in the experimental medium. Spermatozoa with permeabilized plasma membranes were obtained by treatment with fiipin (Carey et al., 1981). The cells were washed twice by centrifugation at 300 X g for 10 mm in NTPC and resuspended in that medium at about 1 X i0 cells/mI. Filipin was added as a 20-mM solution in dimethylformamide (DMF) to 0.15 ml of sperm suspension at a final concentration of 0.3 mM. After 10 mm incubation at room temperature, the sperm suspension was diluted by addition of 0.85 ml NTPC. The cells were recovered and washed once with NTPC by centrifugation as described above. The final sperm suspension was in 0.10 ml of ice-chilled NTPC; the sperm were kept at 0#{176}C in ice until used. These cells are designated filipin-treated mouse epididymal sperm (FTMES). Aerobic Incubation of Spermatozoa

Spontaneous lipid peroxidation was induced by exposure of the spermatozoa to 02 during aerobic incubation. The stock solution of spermatozoa was diluted 3-fold to give a sperm suspension containing 0.2-1.0 X i0 cells/mI; 0.3 ml of suspension was placed in widemouthed specimen bottles (55 X 28 mm) held in a shaking water bath at 3 7#{176}C. The caps of the bottles had liners made of Teflon which prevented contamination of the samples and provided a seal tight enough to prevent loss of malonaldehyde by volatilization from the suspension.

Determination by Malonaldehyde

of Lipid Peroxidation Production

The determination of malonaldehyde was carried out by using a slight modification of the method used previously (Alvarez and Storey, 1982). To 0.3 ml of sperm suspension was added 0.15 ml 40% trichloroacetic acid (TCA) and 0.1S ml of the chosen medium, after incubation was terminated by chilling in ice. The diluted suspension was centrifuged at 2500 X g for 10

mm;
(0.14

the

supernatant

was

added

to

0.15

ml

2%

(w/v)

M) thiobarbituric followed by dropwise

acid addition

(TBA) in distilled water of SN NaOH to give a

clear solution. The rest of the procedure was carried out exactly as described by Alvarez and Storey (1982).

Motility

Assay

Epididymal mouse spermatozoa suspensions were prepared from mature Swiss Webster mice. Excised caudae epididymides of the designated number of mice (8 to 11) were minced into 2 ml of specified medium and the sperm were allowed to disperse for 15 mm at 25#{176}C.Particulate matter was then removed. Duplicate aliquots of 5 .sI of sperm suspension were removed, diluted to 1 ml with distilled water and the number of sperm determined by hemocytometer. The final cell concentration in the stock suspension

Sperm motility was estimated by the method used in previous studies (Alvarez and Storey, 1982, 1983a) in which the percentage motility in duplicate aliquots of the sperm suspension was estimated by microscopic examination and averaged.

Determination

of Rate

of H2

#{176}2

Generation

The formation of H2O2 in the sperm suspension was followed by measuring the oxidation of acetylated ferrocytochrome c catalyzed by cytochrome c peroxidase at room temperature (21-23#{176}C) with a DW-2A dual wavelength spectrophotometer, as described by Holland and Storey (1981).

PEROXIDE

REACTIONS

IN MOUSE

SPERM

835

Determination and Superoxidase

of Rate 0102 Dismutase

Generation Activity

U) -J
-J

a)

Ui The formation of O2 in the sperm suspension was monitored by the reduction of acetylated ferricytochrome c (Azzi et al., 1975) as described by Holland et al. (1982). The activity of superoxide dismutase (SOD) was determined by inhibition of the above

reduction reaction (McCord

added system sperm, as O2 using generator the

and Fridovich, 1969) by

oxidase 1982).

xanthine/xanthine (Holland et al.,

aso

Glutathione
Activities, Both mouse

Peroxidase
and Glutathione enzymatic epididymal

and
activities spermatozoa

Reductase
Content were determined permeabilized in with

filipin (FTMES). In this series of determinations, the cells were not recovered by centrifugation and washed after dilution of the suspension to terminate the 10-mm incubation. In this way, loss of g!utathione from the suspension was prevented. Glutathione peroxidase was assayed by the coupled enzyme method utilizing excess glutathione reductase, which couples oxidation of NADPH to the reaction of the peroxidase with hydroperoxide and reduced glutathione (Paglia and Valentine, 1967). The reaction conditions used were those described for assay of this enzyme in bovine semen by Smith et al. (1979), using exogenous (60 sg protein/mI) reductase and 1 mM cumene hydroperoxide to elicit maximal activity of the enzyme (Lawrence and Burk, 1976). The oxidation of NADPH was monitored by the absorbance change at 365-395 nm using the DW-2A dual wavelength spectrophotometer. Glutathione reductase was assayed in the same system using 5 mM glutathione disulfide as substrate. The activities of the two enzymes were linear with cell concentration over the range 0.7 to 1.7 X 101 cells/mI. Total endogenous glutsthione was estimated by pretreating FTMES with exogenous glutathione peroxidase (2.5 g protein/rn!) and 1 mM cumene hydroperoxide for 5 mm to convert any reduced glutathione to its disulfide. The peroxidase was then inhibited by addition of 0.05 mM ZnCI2 (Splittberger and Tappel, 1979). The glutathione disulfide was determined by addition of 0.2 mM NADPH, followed by glutathione reductase (60 g protein/mi). The absorbance change due to NADPH oxidation on addition of the reductase was monitored at 365-395 nm as described above. Addition of NADPH and glutathione reductase in the absence of peroxidase pretreatment gave the intrinisic glutathione disulfide. Glutathione was calculated by difference.

ii

0 AEROBIC

10 INCUBATION TIME

20

IN HOURS

AT 37#{176}C

b)

0
0/

INERT

50 SPERMATOZOA

100

RESULTS
When NTPC spermatozoa with la), incubation with a net at incubated aerobically in medium

3 7#{176}C, intact
produced time rate over of

mouse
malonaldehyde a period 0.045 of nmol/h

epididymal
linearly 20 h (Fig. per 108

FIG. 1. Malonaldehyde production and mouse sperm motility. Malonaldehyde production by mouse epididymal spermatozoa as a function of aerobic incubation time at 37#{176}C in medium NTPC. The composition of the medium, the sampling procedure,and the spectrophotometric method for determination of malonaldehyde are described in Materials and Methods. Sperm concentration ranged between 0.6 and 3 X 101 cells/mi. Each point represents the mean of 5 experiments; error bars are the standard deviations. The increase in malonaldehyde from 0-20 h can be represented by a linear regression equation through the origin of the form y0.0453x (r=0.993). b) Correlation between malonaldehyde production and percent inert spermatozoa during aerobic incubation at 37#{176}C in medium NTPC. The experimental conditions were those described for Fig. la. Each point represents the mean of 5 experiments; error bars are the standard deviations. The linear regression equation calculated through the origin has the form y=0.0078x (r=0.996).

cells calculated from the incubation. The maximal

medium was 1.2 nmo!/108

initial 20 h of aerobic accumulation in the

cells. The linear

increase
represented and its

of
a slow

ma!onaldehyde
balance removal between through

in
its further

the

medium
reaction,

production

as was also shown for rabbit sperm (Alvarez and Storey, 1982). The nature of the malonaldehydeconsuming reactions was not examined. Mouse

836

ALVAREZ

AND

STOREY

100
time

The decline

effect of mouse

of

medium sperm

composition forward motility

on

the with

was
to value the other

further
zero of 70%

examined.
in a period forward addition mM EDTA

Motility
of motility of to 15 TNC mM 4 h

in
with (Fig.

TNC
an 2).

declined
>-

initial On

I-

hand, and 0.4

sodium (TNCPE) in sperm which 2). CM, the initially, cell

phosphate resulted motility, declined When which in

a significant with an 10% were initial over

enhancement value a period in 90% motility over was a period added in consistent sperm 1983b). added in BSA, to TNC this loss of of 90%

to cells contained

9 h (Fig. medium of

incubated 2% BSA,

5#{176}

population which h (Fig. declined 2).

showed to When a marked observed of BSA and on Storey, when (Fig.

forward

5%
BSA reduction 2),

of to of NTPC motili-

10

(NTPCA), ty effect was

with respect however, to

the

rabbit

4 0

(Alvarez had TNCA. The

NJ

little

effect

give

linear

correlation

between

malona!-

dehyde remained

production valid for all

in in of found as that 0.8 for time the inert figure was same

and
the

cessation
media for

of tested,
the media

motility despite
sperm (Fig. to

50

the become The cells

difference

required different nmol this

3). was
of

malonaldehyde/108 correlation, in NTPC. which Rates

0 0
AEROBIC IN HOURS

the

obtained

5
INCUBATION AT TIME 37#{176}C

10
(I)

FIG. 2. Sperm motility as a function of aerobic incubation time at 37#{176}C in media NTPC (.--s) NTPCA (O--O), and CM (#{149}--#{149}) (upper panel); TNC (0--c), TNCA (Q--O) and TNCPE (0--o) (lower panel). The compositions of the media and the assay of motility are described in Materials and Methods. The experimental conditions were those given for Fig. is. Each point represents the mean of 5 experiments; error bars are the standard deviations.

-J
0 0

Lii

0
0

E
C

Ui oO.5 Ui a
-J
>I

4
epididymal showed decreased When assayed percent correlation tion yields net during inert was the cells corresponding of sperm: an to spermatozoa initial 50% 90% over in forward a period medium motility of 10 NTPC which h (Fig. 2). was to a the

z
4

0 0 % INERT 50 SPERMATOZOA

malonaldehyde this sperm obtained figure as the to period in a and given

production compared time, ib). 0.8 of This nmol lipid

too

linear

(Fig. of index a the

correlamalona!peroxidainert

dehyde/108 tion population

FIG. 3. Correlation between rnalonaldehyde production and percent inert spermatozoa in media NTPC (.--.), TNC (0--c), CM (#{149}--#{149}) and TNCPE (o--o) during aerobic incubation at 37#{176}C.The experimental conditions were those given for Figs. 1 and 2. Each point represents the mean of 5 experiments. The linear regression equation calculated through the origin has the form y=0.00815x (r=O.985).

completely lipoperoxidative

lethal

end

point.

PEROXIDE

REACTIONS

IN MOUSE

SPERM

837

malona!dehyde tested are

difference (NTPCA

shown
in compared

production in Table
rates to TNC),

in 1.
was

the The
nearly but

six media maximum

20-fold

100

these

the

points

Ui -J I0
50

obtained tion of sperm

in all media Fig. 3. The depended on

fell loss the

on the linear correlaof motility in mouse extent of lipid peroxjust as and K of the rate, sperm (Alvarez reported,

idation and was found Storey,

As we

was independent with rabbit previously in a rapid an

(Alvarez

1982). have

high

concentrations produced lated tion

high

the extracellular loss in motility rate loss

Storey,

medium which correperoxidamedia since (Storey epididymal progressively extracellular decrease with this was choline and
tion 130

with compared
Na

increased to the
and

of lipid seen of Na+ in

1982);

0 0 [K]
FIG. 4. Sperm motility after time at 37#{176}C as a function mM

50 mM

tOO

not could

due

to a specific be substituted 1983b).

effect for When

Na+ mouse

Alvarez,

spermatozoa were exposed to higher K+ concentrations in the medium, the result was a steep mouse sperm peroxidation
2-5

in media perimental 1, 2 and periments.

of

3 h aerobic incubaof K+ concentration corresponding to NTPC, from 0 mM K, Na to 100 mM K, 30 mM Na. The exconditions were those described for Figs. 3. Each point represents the mean of 2 ex-

motility within optimal

media (Fig.

and the low

4).

increase same time K+ concentration

in lipid period. of
of

Mouse sperm require a

mM high Na for in

maintenance
The

of motility
dependence

confirmed completely assayed linearly epididymal

by

adding abolished

exogenous the production concentration spermatozoa. did

SOD reduction. not

which The

motility
shown in

of
Fig.

K+
4,

concentration was
very

in
similar

the
to

medium,
that ob-

served
1982).

with

rabbit

sperm

(Alvarez

and

Storey, protime
as

rate of O2 with cell mouse

increase in intact the as of of a

Instead,

Intact mouse epididyma! duced 02 at a rate that (data not reductant shown). The of acetylated

spermatozoa was identity

ferricytochrome linear with O2

concentration was previously spermatozoa this concentration

dependence observed (Holland et

was hyperbolic, with rabbit epididymal a!., 1982). by Analysis means

of

the

c was

dependence

TABLE 1. Rate of lipid peroxidation malonaldehyde production in epididymal matozoa under aerobic incubation at media.a

as measured

by

double reciprocal plot (Fig. 5a) yielded the intrinsic rate of 02 production, vint, and the rate constant K for the reaction of O2 with the rocal 1.96 matozoa. cells. From the 5a), per intercept x 10
10_8

mouse sper37#{176}C in various

slope

of

the

double

recipto be spera K5

plot (Fig. nmol/min The 19.1 of to et al., in mouse

vint was 10#{176} cells of the (cells/mlY cyanide, sperm the

calculated for mouse plot yielded

Mediumb

Ma!onaldehyde (nmol/h per

i0

production cells)

rate

value of presence inhibitory (Holland 02 creased, dence reciprocal

mM 1982),

min1 . In the a concentration SOD activity of independouble of 3.80 108 of of of x of production spermatozoa hyperbolic The

CM

0.0810

NTPC
NTPCA TNC TNCA TNCPE aMalonaldehyde

0.0390
0.0156 0.2800 0.2200 0.0750 was

0.020 0.010 0.006

rabbit

Cu-Zn rate

epididymal

0.040
0.030 0.015 by the thiobarbiValues Methods.

while retaining its on cell concentration. plot (not per min1 in should obtained shown)
108

in

the

presence

determined

tune assay described in Materials and Methods. are the means SD of three determinations. bFor compositions see Materials and NTPCA and TNCA contained BSA at 10 mg/mI.

cyanide nmol/min

yielded cells and

values 19.7

(cells/mi1) The increase cyanide

for vint and K5, respectively. observed in the presence correspond to the loss

838

ALVAREZ

AND

STOREY

20

a)

indirect (1969)
al. dismutase cells,

method
as applied The activity with a to

of

rabbit

McCord sperm

and by
was

Fridovich

Holland
superoxide 3.2 cells U/108

et

(1982).

cyanide-sensitive observed 0.34

E
C

remaining

U/b8

not

E
-.

sensitive
corresponds

to
in the 1973).

cyanide
to the

inhibition.
Mn-containing

The
(Weisiger

latter
enzyme and

tO

located Fridovich, Mouse

H2O2

mitochondria

epididymal at a rate that

spermatozoa was linear

produced with time.

0 >

Exogenous confirming
of acetylated

catalase the

abolished

H2 02

production,

identity
ferrocytochrome c peroxidase.

of H2 02 as the oxidant c in the presence

As in

0 20 (I08 CELLS/mi ) 40

of

cytochrome generation,
a hyperbolic This were at

the

case

of

02
had tion. cells

the

rate
would

of

dependence

H2 02 production on cell concentrabe in expected a second to if the order

dependence to a react rate with

H2 02

40

b)

reaction

significant

compared

the the
assay

peroxidase-catalyzed
acetylated ferrocytochrome production, O2 et by and a!., as

reaction
was found

of H2 02 c
used to

with
to

H202 with
(Holland dence relation (Holland Fig. the 0.95 the 5b. vint

be
this

the

case c

acetylated 1982). of for 1982) the the the Analysis

ferricytochrome of depenplot with plot plot shown (Fig. 5b), 02

1;
C

E 20

means derived et From calculated a!.,

double-reciprocal data obtained the of the

yielded slope for

in

H2O2
value

production
The of intercept 12.1 X

was
of 10_8

nmol/min slope yielded

per a

i#{248} cells. K5

0 0 20 (108 CELLS/mII
FIG. function didymal 5. Rate of O and H2O2 production as a of cell concentration by intact mouse epispermatozoa presented in the form of a double reciprocal plot, a) For O2, the assay was carried out in medium NTPC containing 80-90 Mmol of acetylated fernicytochrome c. The linear regression equation calculated for the double reciprocal plot has

(cells/ml

min1.
at concentrations et in al.,

In
1960),

the
the

presence
inhibitory rate as of

of
to

aminocatalase

40

triazole (Margoliash of H2 02

production measured by

intact

spermatozoa

the
did ing activity, (Mann,

oxidation
not that show these as 1964). do

of
any

acetylated
appreciable lack

ferrocytochrome
increase, intracellular suggestcatalase spermatozoa

cells other

mammalian

The reductase given

glutathione activities in Table 2. The

peroxidase of mouse peroxidase

and

glutathione are given activity

the
assay 90-9

form

y=0.51x + 1.4 (r=0.994). b) For was carried out in medium NTPC 5 MM of acetylated ferrocytochrome c peroxidase. The linear for the double reciprocal + 2.5 (r=0.997).

H202, the containing c and 0.25 regression plot was

spermatozoa

MM cytochrome equation calculated the form y=1.05x

is the maximal hydroperoxide. contents fide. of Glutathione

one assayed Also given in and peroxidase and the glutathione

with cumene Table 2 are the glutathione is quite disulspecific is equally (Chance et enzymes GSH content the in plus free

glutathione

for glutathione specific for the

activity of the Cu-Zn SOD in mouse sperm

reductase disulfide

al., 1979). determining GSSG, of methods of interferences (Li,

The the the cell

use of these two total glutathione, should intrinsic give to the

sensitive to this directly, insensitive

cyanide inhibition. the cyanide-sensitive SOD activities were

In order to test and cyanideassayed by the

chemical

1975).

PEROXIDE

REACTIONS

IN MOUSE

SPERM

839

TABLE mouse

2. Activities sperm treated

of glutathione reductase, with filipin (FTMES).a

glutathione

peroxidase

and

glutathione

content

of

epididymal

Enzyme Enzyme (Mmol/min

activity per 10 cells)

Glutathione NADPH NADH Glutathione

reductase 0.120 0.047 0.200 Content (nmol/105

peroxidase

0.090 0.004 0.100

(4) (3) (3)

Glutathione

cells)

Total

glutathione
disu!fide as GSHb

Glutathione Glutathione aValues bDisu!fide CCalculated

96.0 37.0 59.0c number of experiments equivalents which shown is twice in parenthesis the molar GSSG

7.2 2.6

(3) (3)

are

the content by

mean

of the given

SD. content.

as GSH

difference.

DISCUSSION

activity. share susceptible and correlation the degree with one to calculated 3.80 cyanide absence: same sperm source

This

is in
per 1.96

good
between cells

agreement
vint in per per the 108 108 for

with
#{176}2

the
of of in its The rabbit

Mouse trait spontaneous of peroxidation in

and common: lipid

rabbit

spermatozoa both peroxidation are linear

difference nmol/min and 1.84 of

presence cells cells. in

nmo!/min nmo!/min H2 02 et a!., was

gives

motility

species
activity,

loss. Spermatozoa also lack catalase

and medium. can produce The both H2O2

from activity,
H2O2 produced

the have
and O2 by

two SOD
in the

identified But found H2O2 sperm 02 are is the a

(Holland

1982).

marked between

quantitative
the in rates the two of

difference
sperm species: O2 mouse

was
and

the
sperm of sperm would nmol/min

production far source more of

be
vint 0.95

attributed
for H2O2 nmol/min to 108 cells a

entirely
production per
Vint

to
108
for

the
by cells,

action
mouse which of of 1.90 SOD

SOD: was

active

H2O2,
of in Table

in this regard. it would be

production, 3. In the

Since useful
expressed absence

to
of

compare
as cyanide, v11,

the
as the

rates
done value

correspond per

02

in

the

absence

TABLE rabbit

and

3. Comparison of mouse spermatozoa

values

of

Vint

for

production

of

in the

presence

and

absence

02 and of cyanide.a

of K5 for

reaction

of

O2

with

the

cells

for

vint

(CN)

(nmol/min

per

10

cells)

K5 (cells/ml)1

min

Rabbit (NTP)

0 10mM 0 10 mM for rabbit spermatozoa are from Alvarez

0.17 1.61 1.96 3.80 and Storey (1983a). The medium

1.2 X iO 19.9X 108 19.1 19.7 is shown X 1O X 1O in parentheses

Mouse
(NTPC) aValues

for each species. For rabbit, the difference between in the presence and absence of CN is 1.44 nmol/min per i0 cells, corresponding to 0.72 nmol/per i0 cells for H202 production, in agreement with the value of 0.7-0.8 found (Holland and Storey, 1981). For mouse, the difference is 1.84 nmol/min per iO cells, corresponding to 0.92 nmol/min per 10 cells for H202 production also in agreement with the value of 0.95 found (this paper).

840

ALVAREZ

AND

STOREY

of vint for rabbit sperm; values differ SOD are the

of

mouse sperm is in the presence which are nearly

10-fold that of cyanide, maximal,

for the still the rate

intrinsic

rate

of

SO a minor
oxidative

production role
damage.

and as an in

so

this

enzyme
protecting

plays
against

enzyme mouse which et a!., from

of

by more than 2-fold. activity and intrinsic quite closely intrinsic O2 matched. production
capacity to

In rabbit sperm, 02 production

The sperm

major appears

protective to be the

enzyme glutathione/glutathione

But in mouse sperm, rate runs far ahead

dismutate it.

the

cells

SOD

reductase/glutathione peroxidase system functions in most somatic cells (Chance 1979). It is in this system that sperm in rabbit and difference.
detectable

Comparison terms
1969)

of

the

dismutation

capacity

yields species. U/b8

Storey,

of enzyme units (McCord and Fridovich, of the cyanide-sensitive SOD activity similar values for the sperm from the two Rabbit sperm were found to have 2.7 cells 1981), of this from activity which one (Holland calculates and 1

mouse Rabbit
glutathione

show sperm
(Li,

the have 1975;

most striking little or no Holland and

Storey, 1981) so that, even though they have low levels of the appropriate enzymatic activities, they still lack the substrate to use them. Mouse sperm have GSH those which contents
the reductase

plus

GSSG

contents for other glutathione. as

alone,

U/b8 For 3.2

U/b8

cells0.53 nmol 02 (mm per mouse sperm, the corresponding U/108 sperm cells cells, have
SOD

108 cells). figure is calculates 1

cells. 108

much higher than mammalian sperm Li (1975) reported

by measured

reported contain

from
nmol

which
O27min

one
per

of glutathione,
reaction

cells=0.58

Mouse
insensitive

0.34
activity,

U/b8 sperm;
not

cells
compared

of cyanideto 0.14

ranging sperm of reaction

disulfide;

from from measures

0.5 dog,

to ram the

1.3 and
with

nmol/108 man.
Table

cells reductase glutathione

2 shows

for

The of

U/b8
activity

for

rabbit
would

this
seem

amount
to have

content have than an observed species. the peroxidase or more

difference

comparison

that

much physiological The values constant for

of

impact. K5, the of 02

second with

order the for as cells,

between

rate are

the mouse spermatozoa disulfide content more tude greater than that these other mammalian of the reductase and order of magnitude

a glutathione order of magniin sperm The from activities are also an greater than

reaction

compared in Table 3. A clear difference the two species is that the K5 value sperm is the same in the absence presence of cyanide, in
rabbit

mouse in the lower

while

K5

is 16-fold

in rabbit sperm have interpreted

found with

the absence the difference sperm as

of cyanide. We in K5 values reflecting membrane the reaction intracellular 95% of

the

those observed in the sperm of the other species (Li, 1975; Holland and Storey, 1981). It seems likely one tive be that mouse sperm depend on the glutathioxidamay of the system for major damage. This a wide variation protection suggests in the against that there

the

reactions of O2 alone in the absence of O2 components intracellular Storey,

rabbit K5

with the plasma of cyanide, and plus when

contributions

with in 1983a).
in

the plasma its presence is The

the

SOD

inhibited (Alvarez near identity of K5

of cyanide and

and
for of

glutathione sperm from knowledge,

and SOD protective systems in different species. To the best of our a systematic examination of this also on react depensperm

sperm

presence

for

mouse suggests sperm

sperm that occurs

(Table the both

3), reaction with

independent of 02 plasma mem-

of with

variation has not yet been carried out. Mouse sperm produce H2O2 and with H2 02, as shown by the hyperbolic dence of measured H2 02 (Fig. reaction order, yielding production Sb). This between so that the the double cell concentration implies that the the of plot could found apparent sperm these seems H2 02, H2 02 cell cells the is second equation (Holland be used with

cyanide, mouse brane and interference

be observed

intracellular components, from intracellular SOD.

with little This would were 02. sperm activity sperm size while mouse induced the high

dependence H2 02 and same form reciprocal

already The have means have than

if the SOD in mouse sperm saturated by internally generated that same rabbit and intracellular larger relative conclude We enzyme sperm. mouse SOD mouse to their that, to by

observation about the that less do the protective rabbit

et a!., 1982) in Fig. 5a for O2 for H2 02 in Fig. 5b. The situation rabbit sperm in regard to the of H2 02 is quite production different. on With

considerably

dependence concentration cells, the reaction to have saturation becoming and first zero order

SOD provides some protection spermatozoa against lipid peroxidation by SO, its capacity is overwhelmed

of H2 02 with the cells kinetics with regard to order with with respect respect to to cell

PEROXIDE

REACTIONS

IN MOUSE

SPERM

841 Reprod.
27:1109-1119.

concentration cells/mi possible (Holland that

above

0.2 and

.iM

H2O2

and 1981).

x
It

i0 is Jones,

spermatozoa.

Biol.

Storey,

these

observed

differences

with

regard
difference damage et a!.,

to
in 1959),

cell
in sperm

reaction
sensitivity from but of of the of in this the two this our

with
to rabbit was not reaction

H2 02
H202-induced and

reflect

the
cell (Wales

mouse

assessed. of

Further H2 02 with

characterization spermatozoa beyond under the investigation

species study; laboratory. it

was

deemed

scope

is currently

R. and Mann, T. (1973). Lipid peroxidation in spermatozoa. Proc. R. Soc. Lond. 8. Biol. Sci. 184:103-107. Jones, R. and Mann, T. (1976). Lipid peroxides in spermatozoa; formation, role of plasmogen, and physiological significance. Proc. R. Soc. Lond. B. Biol. Sci. 193:317-333. Jones, R. and Mann, T. (1977). Damage to ram spermatozoa by peroxidation of endogenous phospholipids. J. Reprod. Fertil. 50:261-268. Jones, R., Mann, T, and Sherins, R. J. (1978). Adverse effects of peroxidized lipid on human spermatozoa. Proc. R. Soc. Lond. B. Biol. Sci. 201: Jones, 413 -41 7. R., Mann, T. and Sherins, R. J. (1979). Oxidative breakdown of lipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil. Steril. Glutathione rat liver.

ACKNOWLEDGMENTS This research was supported by NIH grants HD1 5842 and HD-06274. We are particularly grateful to Prof. C. P. Lee for providing a generous sample of purified cytochrome c peroxidase. We thank Mrs. Dorothy Rivers, Mr. Jack Chao, and Mr. Marc Gorman for technical and Ms. Neisha Son for secretarial assistance. REFERENCES Alvarez, G. and Storey, B. T.(1982). Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: its effects on sperm motility. Biol. Reprod. 27:1102-1108. Alvarez, J. G. and Storey, B. T. (1983a). The role of superoxide dismutase in protecting rabbit. spermatozoa from 02 toxicity due to lipid peroxidaBiol. Reprod. 28:1129-1136. Alvarez, J. G. and Storey, B. T. (1983b). Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility. Biol. Reprod. 29: 548-55 5. Alvarez, J. G., Holland, M. K. and Storey, B. T. (1983). Spontaneous lipid peroxidation in rabbit spermatozoa: a useful model for the reaction of 0 metabolites with single cells. In: Oxygen Transport to Tissue-V (D. W. Lubbers, H. Acker, T. K. Goldstick and E. Leninger-Follert, eds.). Plenum PubI., New York, pp. 433-446. Azzi, A., Montecucco, C. and Richter, C. (1975). The use of acetylated ferricytochrome c for the detection of superoxide radicals produced in biological membranes. Biochem. Biophys. Res. Com. 65:597-603. Carey, J. E., Olds-Clarke, P. and Storey. B. T. (1981). Oxidative metabolism of spermatozoa from inbred and random bred mice. J. Exp. Zoo!. 216:285-292. Chance, B., Sies, H. and Boveris, A. (1979). Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527-605. Heffner, L. J. and Storey. B. T. (1981). The role of calcium in maintaining motility in mouse spermaJ. M. metabolism tion of spermatozoa. Holland, M. (1982). Holland, tozoa. Exp. Zool. 218:427-434. K. and Storey, B. T. (1981). Oxygen of mammalian spermatozoa. Generahydrogen peroxide by rabbit epididymal Biochem. J. 198:273-280. K., Alvarez, J. G. and Storey, B. T. Production of superoxide and activity of tion. J.

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