BIOLOGY
OF
REPROI)UCTION
228-239
13,
Localization
of
Piece
HAROLD
Zinc
in
a Dense
Fiber-Connecting
Fraction
of Rat Sperm
Chemically
to Hair
I. CALVIN,
FRANCES
International
Institute
Columbia
U.-F.
for
University,
the
HWANG
Study
College
York,
New
Tails Analogous
Keratin
and
UERMA
of Human
WOHLRAB
Reproduction
of Physicians
NY 10032
and
Surgeons
ABSTRACT
The
intracellular
distribution
of zinc
in mature
rat
sperm
was examined
by subcellular
fractionation
and atomic
absorption
spectrophotometry.
The results
indicate
that >90 percent
of
this trace metal
is located
in the tail, Ca. 85 percent
in -S-S- crosslinked
tail structures
which
do not
disintegrate
in 1 percent
sodium
dodecyl
sulfate
(SDS).
Association
of zinc with -SH is supported
by
the
primary
localization
of
the
latter
within
the
same
SDS-insoluble
material.
The
concentrations
of both zinc and -SH decrease
in the heads and tails of rat sperm
during
passage
through
the epididymis.
Of the minor
fraction
of zinc in the rat sperm
head (5-10
percent),
>75
percent
is not extracted
with SDS and therefore
appears
also to be associated
with
-S-S- bonded
structures.
Over
75 percent
of the zinc in isolated
tails is retained
by a subfraction
which
consists
mainly
of dense fibers,
with connecting
pieces present
as a minor
constituent,
and is largely
non-dialyzable
upon
solubilization
of these
structures.
The
major
component
isolated
from
the solubilized
product,
following
aminoethylation,
is of 35,000
mol. wt. Polyacrylamide
gel electrophoresis
in
SDS also reveals
significant
components
of Ca. 75,000,
25,000
and 15,000
mol.
wt. and trace
components
of 90,000,
70,000
and 50,000
moi wt. The amino
acid composition
of this protein
mixture
includes
11-12
percent
cysteine
and is found
to be strikingly
similar
to the overall
compositions
of wool
and guinea
pig hair,
which
also possess
sizeable
contents
of stably-bound
zinc. The possibilities
that the proteins
of hair and sperm
keratin
(i.e.-kerateines)
share a common
phylogenetic
origin
and undergo
similar
interactions
with zinc during
macromolecular
assembly
are
therefore
noted.
associated
INTRODUCTION
with
the
sperm
(Gunn
and
Gould,
1970).
A
be
keratin,
insoluble
of
according
as “a
defined
by
its
in
spermatozoa
fore
(Zittle
be
element
tions
of
Mercer
the
presence
and
tails
of
has
been
recognized
and
related
present
O’Dell,
1941).
within
keratogenesis
essential
for
Mann,
1964,
wood,
1971),
significance
in relatively
keratins
and
(see
Underwood,
It
may
zinc,
high
necessary
Evidence
of
some
of
such
major
in
1971),
spermatogenesis
in mammals
Gunn
and
Gould,
1970;
during
which
it becomes
fact,
many
zinc
there-
located
normal
is also
(see
Underfirmly
into
of
is
and
tail
1975),
and
form
Calvin,
sperm
from
epididymis,
Gould,
groups
1974)
keratins.
228
in
1970;
its
concur-
tail,
where
al.,
et
sperm
1960;
most
are
Calvin
-S-S-
associated
a
of
(Bedford
release
of
maturation
zinc
1973;
variety
bonding
Following
testis,
during
contents
of
1961;
of the
et al.,
contains
by
1974).
the
1966),
the
Similarly,
rat
which
stabilized
spermiogenesis
(Millar
(Nelson,
dur-
fully-differentiated
the
1974).
Bleau,
groups
the
structures
Accepted
June 4, 1975.
Received
April
22, 1975.
‘In
the
present
context
this
definition
will
be
adhered
to,
and
the
term
kerateine
(Goddard
and
Michaelis,
1935)
will
be employed
to designate
the
cysteine-rich
molecules
which
become
cross-linked
to
in
of
of
of
occurs
et al.,
concentrated
sulthydryl
Calvin,
stages
zinc
with
incorporation
spermatid
Parizek
a
is,
(i.e.-kerati-
association
rat,
elaboration
the
Calvin
the
advanced
the
sperm
linked
in
developing
1958;
most
-S-S-
In
that
sperm
mammalian
largely
most
with
recently
in
within
the
rent
with
zinc
groups.
ing the
(Wetterdal,
a trace
accumulated
of
structures,
sulfhydryl
concentrafor
has
fraction
nous)
mammalian
for
that
may
rendered
crosslinking
The
heads
(1969),
of proteins
disulfide
components.”
complexes
years
the
to
mixture
the
in the
(Gunn
and
Calvin
and
Bleau,
1974)
and
-SU
et aI., 1973;
Calvin
and
Bleau,
(Calvin
both
decrease,
as the
latter
are
oxidized
ZINC
to
form
-S-S-
1971).
In
crosslinks
mature
oxidation
and
(Calvin
et
stable
of
presumably
with
of
with
is
cell
that
only
its
zinc
can
1974),
(Saito
of
al.,
with
Ag+
which
1969),
is
a
be
unless
iodoacetamide,
(Calvin
react
sperm
Bleau,
preferentially
probable
rat
its
association
sperm
with
-SH
localization
This
notion
that
relatively
sperm
within
is
extraction
and
to
which
are
binding
a
preliminary
(Calvin,
1975),
1974),
acid
zinc-binding
bull
keratin
sperm
latter
tails
product,
nantly
of
percent
cysteine,
the
structural
fraction
of
with
heterogeneity
a higher
reveals
determined
to
keratins
Its
percentage
a
compo6
and
19
major
the
zinc-binding
confirmed
and
to protein
composi-
close
homology
with
its
the
of hair.
METHODS
Samples
Sperm
were expressed
cauda
epididymides
of
at 0-5#{176}C from
the caput
400-600
g Sprague-Dawley
and
Tails
Concentrations
Concentrations
of sonicated
sperm,
sperm
heads or
sperm
tails,
suspended
in Buffer
TSA,
were
determined
by dilution
of aliquots
into
0.02
M Tris-HCI
(pH
7.5)
- 0.15
M NaCI
0.02
percent
SDS,
at
concentrations
between
1.5 and 2.5 X 106/ml,
and
counting
with
a hemocytometer.
Scoring
was
performed
as described
elsewhere
(Calvin
et al., 1973;
Calvin,
in press).
Extraction
AND
Heads
operations
were
performed
at 0-5#{176}C. Sperm
were
decapitated
by sonication
in Buffer
P (Calvin
et
al., 1973)
and the heads separated
from
the tails on
sucrose
density
gradients,
which
contained
13 ml each
of: 1.80 M sucrose
(sample
layer),
2.05 M sucrose,
and
2.20
M sucrose.
The sample
layer
was made
up by
mixing
sperm
suspension
in Buffer
P with
a suitable
volume
of 2.20 M sucrose.
All sucrose
solutions
were
prepared
gravimetrically
and included
Buffer
P. Centrifugation
was carried
out in nitrocellulose
tubes with
a Beckman
L3-40
centrifuge,
equipped
with an SW-27
rotor,
at 22,500
rpm (67,000
g, ray) for 60 mm. Tails
were
recovered
from
the
1.80
M/2.05
M sucrose
interface
and heads from
the bottom
of the gradient.
The tail fraction
was diluted
in Buffer
P and concentrated
by recentrifugation
at 67,000
g for 20 mm.
Additional
details
concerning
this gradient
procedure,
modified
from
that described
previously
(Calvin
et al.,
1973),
will be published
elsewhere
(Calvin,
in press).
In some experiments,
it was necessary
to obtain
heads
or tails
essentially
free
of cross-contamination.
For
this purpose,
the isolated
fractions
were resuspended
in Buffer
P and recentrifuged
in the above gradients
(double
gradient
purification).
To assure even suspension
for
subsequent
quantitative
experiments
and
minimize
losses on test tube walls, heads and tails were
resuspended
by brief
sonication
at 55 w in a buffer
with
mild
detergent
properties:
0.02
M Tris-HCI,
pH
7.5 - 0.15 M NaCI - 0.2 mg/ml
bovine
serum
albumin
(Buffer
TSA).
To further
minimize
surface
adsorption,
head fractions
were kept in small nitrocellulose
tubes
(Calvin,
in press).
Cell
ultra-
acid analysis,
which
of cysteine
than
that
(Calvin,
1975),
contin-
previously
suggest
partially
amino
MATERIALS
Sperm
is
respect
is examined.
The
predomi-
identified.
the
sperm
(1973).
been
communication,
identity
rat
A
from
protein
respectively
been
wool.
isolated
al.
has
amino
of
consists
major
have
present
in
et
likewise
contain
overall
been
Baccetti
percent
fraction
connecting
and
also
two
laboratory
a tail
an
zinc-
furnished
70
that
fibers,
and
which
ues
by
dense
nents,
In
has
which
fractionated,
tion
this
over
to
is
bonds.
the
been
from
assigned
resembling
composition
of
has
in mature
rat sperm
of
dense
fibers
whose
proteins
display
pieces,
-S-S-
site
sperm
report
which
structures
by
the
by
(SDS)
of
sperm
tightly
which
or
neither
those
in rat
mature
sulfate
zinc
comprised
from
sonication
on
moiety
of the
is released
cross-linked
zinc
with
observations
dodecyl
information
the
structures.
by
sodium
Bleau,
of
bonded
moderate
disintegrate
Further
by
-S-S-
zinc
by
with
(Calvin
able
most
of
All
with
is consistent
strengthened
little
either
of
groups
229
and
Separation
and
groups.
The
in
et
treated
or
all
rat
endogenous
EDTA
first
in
natural
demonstration
p-mercuribenzoate
-SH
forming
The
the
of
the
groups.
complexes
fraction
removed
these
sulfhydryls
by
KERATIN
M NaH2 P04, buffered
to pH 6.0 (Buffer
freed
of debris
by filtration
through
nylon
mesh
and
low
speed
centrifugation
(Calvin
et al.,
1973).
The
designations
immature
or mature
refer
respectively
to sperm
derived
from the caput
or cauda
of the epididymis.
Sperm
suspended
in Buffer
P were
either
sonicated
directly
in this
medium
for
the
separation
of heads
and tails,
or were
centrifuged
at
1500
g for
10 mm and resuspended
in other
appropriate
media
for the procedures
used in this study.
It
has been
found
that
intact
sperm
retain
the same
amount
of zinc in Buffer
P as in isotonic
media
(Calvin and Bleau,
1974).
P),
of
such
by
minor
sperm
FIBER
rats into 0.02
Bedford,
oxidation
for when added in
can
inhibit
both
element
1973),
complexes
supported
by zinc,
DENSE
SPERM
and
further
alkylation
al.,
occurrence
(Calvin
sperm,
-SH may be retarded
vitro,
this
trace
IN
or
and
Assay
of
Zinc
Suspensions
of sonicated
sperm,
sperm
heads or
sperm
tails in Buffer
TSA were assayed
for total
zinc
by mixture
of an aliquot
with an equal volume
of 2 M
HCI, resonication
at 55 w for 30 sec, incubation
for at
least 1 h, and atomic
absorption
spectrophotometry
of
the supernatant
obtained
by centrifugation
at 1500
g
for
10 mm
(Calvin
and
Bleau,
1974).
In selected
230
CALVIN
experiments,
the completeness
of extraction
with
1 M
HCI was verified
by solution
of the sperm
residue
in
30 percent
(wlw)
HNO3
- 35 percent
(w/w)
HCIO4.
Further
localization
of
the
zinc
was achieved
by
treatment
of intact
sperm,
sperm
heads or sperm
tails
(50-100
X 106/ml)
with
SDS, in some cases supplemented
with
a low
concentration
of dithiothreitol
(DT’f)
and mild
sonication.
SDS
extraction
was
performed
by incubation
in 1 percent
SDS - 0.05 M
Tris-HCI,
pH 7.5 at 25 C for 15 mm and centrifugation
(Sorvall
(rmax).
RB2
centrifuge,
HB4
rotor)
at 10,000
g
SDS-DTT
sonication
was performed
by overnight incubation
of sperm or sperm fractions
at 25#{176}C
in 1 percent
SDS - 0.2 mM DTT
0.05 M Tris-HCI,
pH 7.5. followed
by sonication
for 2 mm at 55 wand
centrifugation
as above.
Zinc contents
of supernatants
and
1 M HCI
extracts
of the
residues
were
then
compared
by atomic
absorption
spectrophotometry,
as described
previously
(Calvin
and Bleau,
1974).
In
two
experiments,
affinity
for
zinc
of sperm
tail
fractions
which
survive
the
above treatments
was
evaluated
by solubilizing
the residues
in 5 M guanidine-HCI
0.01
M DTT
- 0.20
M Tris-HCI,
pH 8.6,
and dialysis
overnight
against
water.
The
total
zinc
content
of the resulting
suspensions
was determined
following
incubation
in 1 M HCI and centrifugation,
by assay of the resulting
supernatants.
Assay
of SH Groups
Duplicate
sets
of
assays
of -SH
in sperm
or
subcellular
fractions
were performed
in quadruplicate,
either with
or without
preincubation
at pH 2. The
suspension
of sperm
or sperm
fraction
(0.1
ml in
Buffer
TSA
at a concentration
of 20-40
X 106/ml)
was either
diluted
with
0.02
M HCI
(0.1
ml)
and
incubated
at 25#{176}Cfor
30 mm,
or else diluted
with
water
and kept in ice. Determination
of -SH was then
carried
out by addition
of 0.3 ml of a solution
which
contained:
50 imoles
of Tris-HCI,
pH 7.5; 0.5 j2moles
of sodium
EDTA;
80 g of bovine
serum
albumin;
and
2.5 pmoles
of (1-’ 4Cl iodoacetamide
(New
England
Nuclear
Corp.),
diluted
to a specific
activity
of 2.90 X
io
dpm/j.imole.
This composition
was modified
for
detection
of the
relatively
low
content
of -SH in
matuze
sperm
heads by increasing
the specific
activity
of the tracer
to 2.90 X 106 dpm/imole,
with
the total
dpm
kept
constant
(i.e.,
only
0.25
moles
of iodoacetamide
present
in each assay).
Following
incubation
for 90 mm at 25#{176}C,1 ml of 10 percent
trichloroacetic
acid
and 0.1 ml of 0.2 percent
bovine
serum
albumin
were
added
and the contents
were
homogenized
by
vortex
mixing,
followed
by brief
sonication
at 55 w. The suspension
was then
collected
ET
AL.
Isolation
and
Fiber-Connecting
Sperm
tails isolated
by double
gradient
purification
were suspended
at concentrations
between
50 and 100
X 106/ml
in 1 percent
SDS - 0.2 mM dithiothreitol
(DTT)
0.05
M Tris-HCI,
pH 7.5, and incubated
at
25#{176}Covernight.
The
suspension
was chilled
to less
than
5#{176}C
for ensuing
procedures.
(Because
of the inclusion
of Tris in the medium,
no precipitation
of SDS
was observed
at this point.)
After
disruption
of the
tails by sonication
at 55 w for 2-3
mm, the surviving
structures
were collected
by centrifugation
at 10,000
g
(rmax).
The
residue
of dense
fibers
and connecting
pieces
was then
washed
at least
twice
with
water
by
resuspension
and recentrifugation
and solubilized
at
25#{176}Cin 5 M guanidine-HC1
- 0.01
M DTT
0.20
M
Tris-HCI
(pH 8.6).
To 10 vol of the dissolved
keratin,
3 vol of 3 M Tris-HCI,
p11 8.6 were
added
and the
proteins
were aminoethylated
at 25#{176}Cby 3 additions
of 0.2
vol ethylenimine
at 10 mm
intervals
(Cole,
1967).
When
-SH was no longer
detectable
with
the
reagent
of ElIman
(1959),
the reaction
mixture
was
cooled
in ice, acidified
with concentrated
HCI to a pH
below
3.0 and dialyzed
against
3 changes
of 0.001
M
HCI (100
vol)
for a total
period
of at least
30 h.
Molecular
weight
distribution
of
proteins
in the
dialyzed
product
was examined
by electrophoresis
in
gels containing
10 percent
polyacrylamide
and 0.1
percent
SDS, according
to Weber
and Osborn
(1969).
The following
standards,
whose
molecular
weights
are
indicated
in
parentheses,
were
electrophoresed
as
references:
rabbit
muscle
phosphorylase
a (94,000),
bovine
serum
albumin
(68,000),
ovalbumin
(43,000),
bovine
s-chymotrypsinogen
A (26,000)
and
horse
heart
cytochrome
c (12,000).
For amino
acid analysis,
samples
were
lyophilized,
dissolved
in 6 M HCI,
equilibrated
with
N2, sealed
under
a vacuum
of less
than
25 z, incubated
for
24 h at l10#{176}C-112#{176}C,
lyophilized
again and analyzed
with a Beckman
Model
120 analyzer
on 1-cm X 50-cm
columns
of Beckman
AA-15
resin,
using the 2-column
system
of Spackman
et al. (1958).
RESULTS
Intracellular
Zinc
in
intracellular
rat
sperm
ingredients
except
1’ Cl iodoacetamide,
before
addition
of the tracer
(2.5 moles/0.1
ml).
Under
these
conditions,
95 percent
inhibition
of 1’ ‘ Cl -incorporation was observed.
been
dealt
from
sperm
and
very
little
of
from
the
sperm
that
(Calvin,
1975;
treatment
rat
bonded
tail.
within
over
by
From
Calvin
-S-S-
spares
bonding
this
it was
sperm
(i.e.
resides
-SH
tail,
also
zinc
in
previous
of
the
zinc
the
tails
It
percent
within
trace
element
by
incubation
and
Bleau,
concluded
primarily
-SU
has
can
in
be
SDS
1974),
that
localized
within
the
within
fraction
is
and
a
only
those
structures
(Calvin
and Bedford,
keratinous)
Similarly,
the
this
of
with
laboratory.
90
is localized
which
stabilized
in
that
this
been reported
in mature
rat
1971).
for 15 mm,
in a
presence
of
all
distribution
has
communications
moles
total
(1959)
in
the
of
-SH
The
released
reagent
0.4
ml
Distribution
and
on a Whatman
GF/C
filter,
which
was washed
and
assayed for radioactivity
as described
previously
(Calvin
et al.,
1973).
Counting
efficiency
was
85-90
percent.
The specificity
of the above assay was tested
with
unfractionated
sperm
by incubation
with
5.0
of ElIman’s
volume
of
Analysis
of Dense
Piece
Fraction
of
zinc
an
the
sperm
predominantly
structures
stable
in
ZINC
SDS
(Calvin,
on
constituents
distribution
rat
following
of
maximize
alkylation
doacetamide,
samples
at
it
was
2
samples
optimal
caput)
for
-SH
inhibition
was
of
able
by
by
Additional
prolonging
37#{176}C was
with
the
largely
and
any
not
to
of
of
-SH
with
be
in
and
of
within
the
TABLE
1. Intracellular
tail
both
Ellman’s
cells
ascribed
been
rat
1973).
In
per
during
the
by
the
primarily
two
experi-
of zinc and
that
only
cated
rat
mm
in
0.1
M
sulfhydryls
by
-SI-I
in rat
Sample
1
Caput
sperm
by
40-50
sperm
Cauda
heads
sperm
Cauda
sperm
headsa
Cauda
Cauda
aThiS
samples
blntact
due
was
sample
was
(see Materials
cauda
washed
incubated
and
sperm
once
sperm
with
they
not
were
react
exposed
proposed
that
acid from
these
1974),
which
has
been
by
Bleau,
with
the
alkylation
iodoacetamide
this,
of
it
was
the
by
incubation
in
zinc
assay
in
sonifor
1 mM
the
al.,
determined
is released
-
et
present
the
7.5,
of
(Calvin
of
pH
a signifi-
did
90
EDTA
-
absence
of
Mmoles/109
cells
SE1
Zn
SI-I/Zn
+
13.7
12.3
1.23
11.1
0.095
11.3
+
1.08
-
1.08
+
5.15
-
3.19
0.76
6.8
0.073
0.047
0.046
1.6
5.04
3.27
0.58
8.7
+
3.89
0.56
6.9
-
3.04
+
4.24
0.56
7.6
-
2.76
‘4C-iodoacetamide
of
10-fold
the
specific
activity
used
for
all of the other
Methods).
were
with
tails
immature
preincubation
+
Cauda
sperm?
SDS-treated
preincuba-
spermatozoa.
+
sperm
of sperm
percent
Tris-HCI,
-
2
stages
that
iodoacetamide
-
Caput
in both
by
It has
and
Acid
Experiment
the
decrease
except
removed
support
of
development
5 mM
respectively
supports
sulfhydryls
acid.
sperm
tail.
indicates
to interfere
sperm
-SH
the
final
unless
inhibitor
is zinc (Calvin
cauda)
zinc varied
per i09
cells.
1)
major
shown
both
alkylation
of
way
in
within
zinc
fractions
iodoacetamide
Mmoles
of
distribution
percentage
5.7
these
is illustrated
cant
with
the
to
-SI-I
all
(Table
heads
zinc
as well,
cells
the
from
epididymis.
of
(i.e.,
of
in
sperm
rat
mature
4.9
2
p1-I
in some
and
groups.
between
localization
obtain-
therefore
-SH
ElI-
incubation
i09 cells, whereas
determinations
between
0.58
and
0.77
j.imoles
The
percent
temperature
inhibitable
not
certainty
ranged
period
the
any
-SH
and
presented
and
also
and caput
sperm
in Table
1. These
mature
during
in
Stimulation
at
-SH
cells
data
-SH
tails
and
tion
the
with
incubation
could
Estimates
sperm
95
the
that
of
primarily
and
maturation
these
incorporation
the
increasing
reagent
by
of
immature
heads
of
is
hardly
fraction
sperm
located
it
where
documented
are
conclusion
of
of
[‘4C]-incorporation
reagent.
for
heads
Under
confirmed
zinc
1,
removes
in immature
that
Comparison
1, this
specificity
also
are
and
reaction
the
SDS
caput
confirm
To
-incorporation
sperm.
conditions,
assay
reagent
ci
except
Table
a minor
Estimates
sperm.
heads
to
Table
in
that
unfractionated
preincubate
in
[
tested,
(i.e.,
immature
man’s
shown
only
and
cauda
L’4Clio-
before
231
quoted
zinc
im-
glass.
by
to
mm
30
stimulated
all
KERATIN
demonstrated
dilute
fractions
to
groups
As
pretreatment
been
of
sperm
necessary
for
since
of
adsorption
of-SH
pH
two
handling
and
by
iodoacetamide.
in
the
sperm
losses
have
these
development
for
minimize
of
sperm
the
procedures
suspensions
FIBER
ments
the
within
repeated,
with
DENSE
1975).
Studies
proved
IN SPERM
buffer
incubated
TSA,
for
15
resuspended
mm
at
25#{176}Cwith
in this
medium
1% SDS
and
-0.05
sonicated
M Tris-l-ICI
as usual.
(p11
7.5).
The
resi-
CALVIN
232
TABLE
2. Stability
of zinc
complexes
ET AL.
in rat sperm.
No.
Source
Fraction
of
Treatmenta
detn.
% Retained
Caput
Whole
sperm
SDS
2
64
Cauda
Whole
sperm
SDS
SDS-DTT,
3
3
92
80
aA
described
at
tion
alone
The
possibility
SDS
SDS, solubilization
SDS-DTT,
sonication
SDS-DTT,
sonication,
solubilization
2
2
4
2
94
72
77
62
I-leads
SDS
2
76
pH
ca.
that
-SI-I
and
Methods.
2, whereas
removes
increases
tional
Tails
in Materials
preincubation
sonication
90
such
of
pretreatment
by
reactivity
mechanisms
is of
the
zinc.
0.01
in
N HCI
alternative
course
the
preincuba-
percent
binding
of
which
tin,
or addi-
not
zinc
Extraction
solubilizes
Linked
The
of Zinc
in -S-S-
Thus,
Structures
within
inability
structures.
of
SDS
to
extract
an
appreci-
fraction
of the zinc from
mature
rat
(Table
1) confirms
its localization
within
able
structures.
As
percent
of
tion
of
by
indicated
the
zinc
cauda
SDS.
On
the
the
zinc
in rat sperm
it
may
be
in the
structures
which
components,
bonds,
percent
is situated
fibrous
In
contrast
to
the
same
structures
crosslinks,
lished
whose
until
Therefore,
percent
in SDS,
as a conse-
deficiency
it
of
is
the
not
zinc
of
reached
caput
exp.
to
1).
gain
contain
present
Nevertheless,
information
only
that
in
the
attempts
concerning
been
sperm
cauda
only
64
was retained
sperm
5-10
has
unfractionated
1974).
following
SDS extraction
(Table
2).
Rat sperm
heads isolated
by double
purification
zinc
originally
of
estab-
the
Calvin,
surprising
in
zinc-bind-
sperm
-S-S-
is not
and
the
rat
mature
partially
have
of
1!I
relative
(Bedford
major
Dense
Assembly
mature
sonication
crosslinked
mito-
in
complement
in the
-S-S-
Calvin,
epididymal
full
these
-S-S-
outer
and
behavior
of
dense
in caput
sperm
epididymidis
their
post-
Bedford,
These
piece,
and
the
of
by
(Bedford
chroma-
of
tail
SDS.
crosslinked
sheath
membranes
disintegrate
of
their
in
the
more
localization
by
2).
SDS,
1971),
25 percent
of the zinc.
zinc in the head is located
of
in
(Table
(Table
within
connecting
1974).
quence
tail
85
and
Piece
material
achieved
5 percent
the
stable
sperm,
sperm
90-9
(Calvin
Zinc
Further
ing
tails
in
are
the
fibers,
chondrial
that
sperm
ca.
are
include
outer
or
90
remnants
or
Fiber-Connecting
extrac-
that
cell
which
over
resides
estimated
zinc
2,
sperm
assumption
1),
total
Table
Protein-Bound
stable
following
epididymal
of
the
in
is retained
sperm
one
head
1 percent
except
and
approximately
most
the
with
all structures
sheath
releases
Localization
in
head
perforatorium
acrosomal
precluded.
material
the
gradient
percent
sperm
have
the
of
the
(Table
been
nature
1,
made
of
FIG.
sucrose
1. Rat sperm
density
gradient
tails isolated
centrifugation.
by
sonication
X330.
and
ZINC
IN SPERM
DENSE
FIBER
KERATIN
tion
at
percent
233
10,000
of
g for
the
consequences
are
illustrated,
The
tails
of
Figs.
are
I and
in
treated
consists
of
2.
connecting
by
far
sperm
Fig.
display
largest
normally
FIG.
SDS
percent
7.5).
and connecting
following
sonication
2. Dense
fibers
centrifugation,
0.2
-
mM
DTT
-
0.05
pieces
isolated
of tails
in 1
M Tris-HC1
(pH
X500.
found
along
although
elements.
ly
mM
after
DTT.
incubation
The
residues
in 1 percent
isolated
SDS
by
-
centrifuga-
0.2
sperm
(Baccetti
with
Similar
and
borders
they
fibers
Other
are
pre-
appears
connecting
their
treatment
of
staining
periphery.
The
are
spots
are
characteristically
of
the
dense
are seen
1973),
of
ab-
fibers,
as detached
intense-
have
a crude
with
fibers
in
integrity,
mottled
peripherally-localized
bodies
al.,
dense
dense
structural
and
at
density
the
et
the
electron-dense
occasionally
dense
1)
2)
been identifibers
and
which
and the
of
fraying
hyperchromatic
following
or tails
small
detectable,
evident
the
(Fig.
(Fig.
component.
loss
the
most
75
amounts.
some
high
tails
and
micrograph
dense fibers
by
of
3),
although
In the electron
3, both the
as indicated
which
are
by
(Fig.
trace
over
residue
fibers
predominant
structures,
piece
the
size. These have
as dense
outer
pieces
in only
sent
way,
fragmented
the
contain
sonicated
When
this
bodies
of uniform
fied
respectively
mm
10
originally
present
(Table
2).
of such disruption
of sperm
first
of all, by comparison
zinc
been
observed
fraction
thiols
or
and
might
of
with
bull
EDTA
therefore
1
3
*
Ia
4
c’
4.
V
‘I
b
FIG.
3. A field
of dense fibers
containing
one connecting
piece (cp). as viewed
in thin section
in the electron
microscope,
following
preparation
as described
previously
(Bedford
and Calvin,
1974),
modified
by substitution
of 0.1 M NaH2 P04,
buffered
to pH 7.4,
for borate
buffer
during
fixation.
Disintegration
of the fibers
is
especially
evident
along
their
periphery
(arrows),
where
prominent
hyperchromatic
bodies
(h) are also seen.
(Courtesy
of Dr. G. W. Cooper.)
X 18,000.
234
CALVIN
involve
some
fiber
form
of
substructure.
rearrangement
The
denatured
sperm
components
to the hyperchromatic
bodies
yet
be excluded.
The affinity
of
of
possibility
sperm
tail
ET AL.
dense
that
other
may
cannot,
contribute
however,
keratins
for
zinc
has been
found
to persist
even in solubilized
material.
When residues
from
SDS extraction
or
SDS-DIT
sonication
of tails were dissolved
in 5
M guanidine-HCI
0.01
M DTT
0.20
M
Tris-HCI
(p1-I 8.6)
and
the proteins
reprecipitated
the
resulting
suspensions
the
majority
of
was
retained
sis
for
zinc
2),
zinc
revealed
in the original
its
and
stable
-
OVALBUMIN
-
CHYMOTRYPSINOGEN
residues
solubilization
supporting
that
a
BSA
-
dialyzed
against
water,
gradually.
Assay of
following
(Table
with
the
PHOSPHORYLASE
dialy-
association
protein.
-
Composition
of the
Connecting
Piece
The
dense
which
Dense
most
of
tail,
is heterogeneous
content.
Following
guanidine-HC1
polyacrylamide
derivative
whose
position
35,000
molecular
components
zinc
SDS
25,000
positions
50,000,
70,000
weight
and
of
diaband
approximately
75,000
and
to
3 faint
molecular
90,000
acid
the
a strong
(Fig. 4). In addition,
3
of
molecular
weights
suggest
and
amino
fraction,
rat sperm
reveals
corresponds
15,000,
piece
in the
electrophoresis
in
whose
The
the
with
respect
to protein
solution
of the material
in
DTT
and
aminoethylation,
gel
lyzed
minor
Fiber-
Fraction
fiber-connecting
retains
-CYTOCHROMEc
are
bands
weights
of
present.
composition
of this
mixture
of proteins
is presented
in Table
3. Resemblance
to the overall
composition
of samples
of
wool
(O’Donnell
guinea
listed
evident.
cysteine,
in
and
hair
pig
the
same
Similarly
serine,
correspondingly
nine, histidine
Thompson,
(Steinert
and
table
for
prominent
proline
and
and
1973),
comparison,
is
percentages
arginine,
as well
of
as
low percentages
and methionine,
coincidental.
Moreover,
the
the hydrophobic
residues,
1962)
Rogers,
of
seem
phenylalamore than
total
percentages
alanine,
isoleucine
of
leucine,
methionine,
phenylalanine,
tyrosine
and valine
in the sperm
keratin
is within
thd
typical
range
of 25-30
percent
suggested
by
these two varieties
of hair.
Finally,
the sperm
sample
yields
a
aspartic
and
glutamic
hydrolysis.
present
The
as amides
similar
total
acid
fraction
has not
percentage
residues
of
been
these
upon
of
acid
originally
determined.
+
FIG. 4. Analysis
of solubilized
dense fiber-connecting piece
fraction
by electrophoresis
in 10 percent
polyacrylamide
gels, containing
0.1 percent
SDS, and
staining with 0.25 percent Coomassie blue. The sample
shown
was run in parallel with standards,
for 2 h in
0.5 cm X 6 cm gels, at a current
of 6 ma per gel.
Preincubation
with
2-mercaptoethanol,
which
did not
affect
the results,
was omitted.
Migration
was towards
the anode
(+).
The distance
from
the origin
(-)
of the
leading
edge
of each
band
was used
to determine
mobility.
The
relative
migration
of 5 proteins
of
known
molecular
weight
(see Materials
and Methods)
is indicated.
BSA = bovine serum albumin.
ZINC
TABLE
3. Amino
analyses
acid
IN SPERM
of rat sperm
tail
DENSE
fibers,
FIBER
wool
KERATIN
and
guinea
235
pig hair.
Residues/100
Amino
acid
Alanine
Arginmne
Aspartic
Cystemnea
acida
acida
Glutamic
Glycmne
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
W00lc
3.1
7.5
9.3
11.6
7.8
6.0
1.1
5.5
6.6
6.5
11.4
11.3
8.8
0.8
3.5
3.4
10.2
6.2
1.0
1.5
8.6
7.8
3.0
0.5
2.9
6.0
9.6
6.1
4.1
5.9
Serine
Threonine
Tyrosmne
Valine
9.4
2.9
5.6
4.7
aBecause
of
the
procedures
employed
for
solubilization
and
between
cysteine
and V2-cystine,
or between
aspartic
and glutamic
bAnalysis
based
on two
24-hr
hydrolyses,
uncorrected
for
yzed
as aminoethylcysteine.
cAdapted
from the
dAdapted
cysteine.
The
data
from
the
trace
content
O’Donnell
of
of
data
and
Steinert
of citrulline
Thompson
and
been
has
the
likely
that
rat
the
and
The
therefore
such
a
of
its
visibly
rial
may
amounts
the
remainder
open
to
zinc
in
of
zinc
in
Besides
two
structures,
namely
the
and
were
rat
(Fig.
dense
The
the
resistant
lower
not
distinguish
residues
as cysteic
analyzed
been
anal-
acid.
as carboxymethyl-
and
Mohri,
to
piece
in
retained
Here
too,
-S-S-
trace
SDS,
the
implies
sonicacon-
of
tions
vivo
for
of
sperm
testis
and
administration
1961),
and
is
further
findings
in
the
bull
and
contain
(Morisawa
of
zinc
in
head (Table
1).
appear
to bind
when
extracted
heads
retain
their
zinc
zinc
percentage
with
approxi-
zinc.
entirely
accord
with
almost
is in
evidence
rat
to
the
structures
sperm
of
rat
urchin
with
linked
percent
1974),
of
is
which
Calvin,
sea
A small
isolated
75
of
the
element,
the
tail
complex,
and
in
1972).
autoradiographic
SDS
that
filament
is associated
tail
percent-
with
concentrations
and
outer
fiber-connecting
(Bedford
shown
sperm
the
axial
SDS
Localization
remain
fibers
the
in
mately
bonded
sheath
are
1974).
3),
mate-
distribution
-S-S-
fibrous
membranes,
Calvin,
the
other
analyzed
zinc in the dense
in comparison
appreciable
both
sperm
residues
with
has
structures.
rat
residues
soluble
Since
of
relative
Cysteine
Cysteine
do
amides.
Cysteine
of
zinc.
In addition,
the minor
fraction
of zinc in rat sperm
tails solubilized
by
1 percent
SDS is probably
associated,
at least in
other
isolated
these
and
in
minor
pieces
the
analyses
centrations
by
isolation
in
present
location
conjecture.
and
visible
a
that
tion
part,
fibers.
underestimations
pieces,
(Bedford
the
only
their
of
connecting
mitochondrial
in
zinc
dense
connecting
during
actually
precise
the
and
insignificant.
and
Figs.
of
readily
these
residue
of SDS extraction
(Table
2),
the two
structures
eliminated
by
in SDS-DTT
may contain
significant
the
dense
2),
Contamination
represent
The
mature
in
the
constitute
recoveries
in
the
(Table
of
although
fibers
damaged
percent
of
4.9
7.6
6.0
10.8
12.6
9.2
0.6
3.2
7.0
2.6
0.6
2.3
7.2
11.3
7.0
3.8
4.8
hydrolysis,
age of
fraction,
percent
tail
primarily
is relatively
dense
75
the
percent
content.
appears
zinc
with
than
preparation,
structures
the
70
pieces,
fraction
of
(1973).
pig
omitted.
it
described
in
consists
Connecting
of
more
present
over
It
fraction
preparation
3 retains
originally
sperm.
here,
is associated
fibers.
zinc
presented
major
spermatozoa
outer
2
results
Guinea
haird
acids and their respective
losses during
hydrolysis.
(1962).
Rogers
DISCUSSION
From
residues
Rat sperm
tail fibersb
obtained
epididymis,
of
65 Zn
with
sec-
following
(Millar
et
supported
(Baccetti
within
earlier
et
in
al.,
by
similar
al.,
1973).
236
CALVIN
Moreover,
kinetic
zinc
uptake
by
Parizek
et
primary
effects
(Millar
togenesis
and
rat
al.,
deprivation
on sperma1960;
Orgebin-Crist
et
predominant
al., 1971)
significant
zinc-binding
for
zinc
in
tail.
On
rat
suggest
that
requirement
spermatogenesis
occurs
during
of the
spermatid,
and may
directly
in differentiation
the
other
with
sections
et
hand,
dithizone
the
staining
therefore
of the
of
rat
has indicated
testis
that
zinc
is primarily
located
in the heads
of testicular
sperm
(Timm
and Schulz,
1966).
It is not clear
at present
to what
extent
this apparent
contradiction
reflects
chemical
existence
of our findings
with
epididymal
unreliability
of dithizone
for the
determination
of zinc,
the
of a loosely-bound
fraction
of
sperm
which
is nearly
absent
testicular
sperm
cytoactual
zinc in
in the
more
mature
cells,
or the removal
of such a
fraction
by suspension
of sperm
in buffer,
as in
the
present
experiments.
reported,
on the
basis
of
analysis
of unwashed
sperm,
human
sperm
are the major
1966).
However,
ing,
using
sperm
washed
zinc-rich
attempts
which
to repeat
had
been
between
head
(Friberg
distribution
of
heads
and
tails
remains
to be established.
Facilitation
of zinc
reagents
has
12
percent
fiber-connecting
that
sperm
1975;
of
release
indicated
of zinc
in rat
groups
(Calvin,
The
the
and Nilsson,
stably-bound
human
by
sperm
sulfhydryl
major
fraction
is associated
with
-SH
Calvin
and Bleau,
1974).
cysteine
content
fraction
(Table
of
the
dense
3), which
con-
firms
earlier
observations
of the high
-SI-I in rat sperm
dense fibers
(Nelson,
consistent
with
this interpretation.
the relatively
low content
other
of the 20 common
binds
zinc with
comparable
al.,
this findcarefully
to remove
contamination
from
the
seminal
plasma,
failed
to detect
zinc in
the human
sperm
1974).
Thus,
the
zinc
It
has also
been
X-ray
microprobe
that the heads of
site of zinc (Hall,
content
of
1960),
is
Moreover,
of histidine,
the only
amino
acids
which
avidity
(Hallman
et
1971;
of
these
3-fold
that
molecular
of zinc
et al.,
Diamond
quantitatively
of
mg
nearly
and
1970;
most
evaluation
per
zinc
1966)
al.,
the
elongation
be involved
developmental
studies
on
testis
(Wetterdal,
1958;
ET AL.
30,000
that
one
this
in
sperm
11-12
percent
ethylation
when
has
the
the
In
rat
a
sperm
of
failed
(15
the
other
hand,
pig
hair
(Steinert
which
fall
into
the
known
as
hard
with
consistent
phylogenetic
of
(e.g.
trace
tissue
dense
pieces
(Baccetti
fibers,
and
which
also
remnants
et al., 1973).
contained
of
connecting
the
fibrous
The concentration
sheath
of
tance
pro-
of
from
rat
contents
glycine
(12
nail,
metal
of
in Table
3,
the overall
and
Rogers,
category
of
stable,
in
hair
similarities
are
of common
relatively
and
promi-
other
moreover,
well
hard
established
1971).
Therefore,
the
here that stable
zinc-sulfhydryl
to
sperm
the
tail
to its role
in other
to
evidence
the
zinc-sulfhydryl
there
-SH
1973),
substances
horn)
and the essential
role
in the formation
of kerati-
-
contrast
sperm
tail,
of whether
discrepancies
of the dense fiber-conisolated
in the present
compositions
of either
Thompson,
1962)
or
zinc
are,
relevance
By
and
the
Price
as illustrated
between
The
origin.
content
linked
of
to
these
keratins.
Such
the
possibility
intrinsic
weight
is 30,000,
has been
preparations
of bull sperm
solubilized
and
acid composition
piece
proteins
and the overall
(O’Donnell
and
are
crude
value.
unchanged
exist
-S-S-
from
this
analyses.
similarities
plexes
molecular
were
evaluate
acid
certain
isolated
fibers
to
amino
whose
mate
increase
percent)
cysteine
approxi-
it
ground.
were
higher
than
those
listed
in the
communication.
Since
details
concernpreparation
are not yet available,
it is
(Underwood,
discussed
percent),
firm
according
et al. (1973).
1972),
favors
zinc ligands
in
of even higher
(19
to
guinea
nous
on
dense
which
communication,
striking
keratins
of this
total
the
isolation
fraction
whose
possible
nent
be
to be
cysteine
However,
in
preliminary
between
amino
necting
study
wool
the
has remained
dense
cysteine
On
Its
has described
of a dense fiber
percent)
present
ing this
not
for
isolated
likely
fraction
estimate
carboxamidomethylated
cedure
of Baccetti
(1973)
weight
the
solubilized
aminoethylated
fraction
to a repeat
sequence
of
in guanidine-HCI
DTT
and amino-
reduction
Likewise,
fibers.
aminoethylated
is most
to
the
the
primary
dense
ligand.
determined.
piece
Giroux
and
Henkin,
cysteine
residues
as the major
this fraction.
A protein
fraction
content
fibers
appears
Submitting
dense
fiber
sperm
estimate
fiber-connecting
predominates
proposed
is the
the
the
major
zinc-binding
content
has not yet been
the
it has been
molecular
dense
was
Since
was
bull
reasoning,
of 35,000
rat
preparations
whole
cell.
component
protein
ligand
component
from
therein,
cysteine-rich
analogous
By
fibrous
of the
weight
evidence
com-
development
structures
of
may
keratinous
for
the
interactions
is no clear indication,
groups
play
a major
be
cells.
imporin
the
as yet,
role
in
ZINC
binding
mature
this
zinc
sperm
trace
within
head,
metal
the
the
IN SPERM
rat sperm
head.
In the
primary
localization
of
within
-S-S-
linked
(i.e.
soluble)
structures
tion
of -SI-I alkylation
(Table
2) and the
by preincubation
(Table
that
1)
suggest
this
However,
assay of -SH
head is not measurably
cubation
content
during
DENSE
is
a
SDS-instimulaat pH 2
possibility.
pH
2, which
tion
of -SH
(Table
1). Furthermore,
the apparent
of
-SH
in this
structure
decreases
largely
greater
and
so that the ratio
from
>10
of -SH
in the
sperm
immature
to
of
mature
head
sperm.
This
developmental
tive
in
levels
of these
with
indicates
decreases
the
present
1964),
in
head,
tail.
-SH
and
zinc
during
maturation
of the
tail may
be interpreted
on the basis of evidence
that:
1) the
great majority
of both
-SH and Zn is located
in
the
tail
(Table
during
maturation
(Calvin
and
1974),
and
is therefore
maturation,
(i.e.
2),
excess
of
ported
of
the
and
the
for
what
1961),
The
are
than
-SH
of
zinc
of
-
interacting
resulting
maturation,
not
same
plexed
most
protected
of
by
time,
with
are
zinc
concomitantly,
available
for
the
oxidation
the
mature
sperm,
and
a significant
zinc-mercaptide
which
contain
percent
decrease
is some1).
been
deduced
equally
stable.
During
groups
which
to -S-S-. At
weakly
released,
are
the
comand
chelates,
great
majority
of
of sulthydryls
the
atoms
most
and
are
in
zinc
exist
stable
1 zinc
as
of
atom
the
from
trace
oxidation,
metal
of
1)
and
regulates
-S-S-
that
the
extent
crosslinking
which
must
mammalian
recently
reac-
(Table
In addition,
it is
shielded
by zinc are
in fact
both
tails
to bind
the
important
It has
copper,
sperm
shown
in
have
(Mann,
competitive-
cations
groups
-SH
have
been
proposed
to
systems,
e.g., the cell membrane
If such antagonism
does occur
in vivo
be
hair
reinforced,
suspected,
studies
in
as a result
sheep,
that
analogy
may
on
on the
such as
occur
in
(Chvapil
other
et al.,
between
Cu2
in the sperm
tail,
and
for
between
and
the
sperm
tail keratins
it has long
been
of nutritional
trace
amounts
deficiency
of Cu2
are necessary
for the normal
oxidation
of -SH
to -S-S- in wool
(Marston,
1946;
Underwood,
1971).
In addition,
the
affinity
of the
-SI-I
groups
in reduced
wool
for
Zn2f
has been
demonstrated
uptake
of
reduction
1974).
the
differential
cation
both
-S-S-
possible
structures
to
by
this
of
The
may be
of the
1974).
groups
effects
of these
two
of labile
functional
differentiation
exposed
sulfhydryls
to disulfides.
Finally,
the
Opposing
oxidation
sulfhydryl-rich
of
is only
is largely
fraction
3 sulfur
to
in
the majority
of -SH
Secondly,
although
the -SH
oxidize
zinc
which
sulfhydryls
3 re(Kagi
zinc in these structures
-SH
groups,
not
all
complexes
in
First of all, certain
-S-Sthe dense outer fibers)
as complexes
the
with
of
(Table
has
proteins
and zinc, in which
is not associated
with
zinc.
all
in both
enhances
the basis of studies
which
indicate
that
Zn2
can inhibit
the oxidation
of rat sperm
-SH to
-S-S- by Cu2
in vitro
(Calvin
et al., 1973).
would
1) is well
maturation
these findings.
structures
(e.g.
nearly
zinc
number
model
assembled
to
(Table
5) the
that
-S-S(Tables
complexes
and
following
explain
linked
of
sperm
epididymal
greater
within
zinc,
a factor
and
resist
ly to human
sperm
(Maynard
et al., 1975),
exert
opposite
effects
on -S-S- crosslinking,
1972).
Zn2
during
structures
coordination
during
those
linked
localized
zinc-mercaptide
Vallee,
-SU
ratio
mature
Calvin,
within
-S-S-
is also
is lost
to -S-S-
and
localized
SDS-insoluble)
4)
immature
Bedford
become
3) zinc
stabilized
-SH which
converted
1971;
which
1 and
2) the
becomes
Bedford,
structures
and
1),
tail,
to
114 Cl iodoacetamide
protected
this
known
consequences
for sperm
motility.
been
suggested
that
zinc
and
respec-
in the
in
the
removes
with
arrangement
a poor
the
constituents
observed
comparable
that
between
two
that
in
least
correlation
contrast
The
at
<2
a far
are
and Vallee,
1961),
it is quite
that
preincubation
of sperm
at
and
Bleau,
that the -SH
therefore
extent
that that of zinc,
to
zinc
has diminished
sperm
to
237
alkylation
(Kagi
understandable
(Calvin
likely
maturation
KERATIN
(Kagi and Vallee,
1961).
Since
such
complexes
sperm
prein-
epididymal
in the immature
affected
by acid
FIBER
bonds
in
of
when
phylogenetic
mammalian
sperm
that
-S-S-
(Bedford
and Calvin,
in zinc
(Martin
et
crosslinked
auxiliary
sperm
if zinc
At
tails,
it would
is concentrated
the
same
time,
be relevant
elements
kinship
to
or hair seems
the
the
keratin-like
may
species,
and Calvin,
possess
Friedman,
during
of
similar
of mention
which
zinc
sperm
mammalian
development
it is worthy
of
and
maturation
occur
in sub-mammalian
vertebrates
(Bedford
sperm,
(Masri
function
and
studies
on the
before
and after
which
including
1974),
ineven
the keratins
of
unlikely.
Thus,
tails
linked
of octopus
outer
fibers
1974),
are unusually
rich
al., 1973).
Wherever
-S-Selements
do
occur
in
be of interest
to establish
in these structures.
it
is recognized
that
the
238
CALVIN
prominence
of
zinc
in
the
axial
filament
plex of sea urchin
sperm
(Morisawa
1972),
in prostatic
and
vesicular
(Mawson
and
Fisher,
nell et
human
al., 1972)
epididymis
1974),
sperm
the stimulatory
metabolism
been
claimed
and
for
al.,
1953;
Mann,
in the
(Schell
zinc
1955;
in
(Fujii
et
1967;
et al.,
Lindholmer
and Eliasson,
1975),
its marked
affinity
the sperm
its ability
membrane
to counteract
Cd2+
on
(Parizek,
the
Gould,
functions
(Blank
the
testicular
1957;
Gunn
1970)
for
effects
on
which
have
1955;
of
species
Saito
et
al.,
1974; Maynard
for proteins
of
et al.,
harmful
1974),
and
effects
of
vasculature
et al., 1963;
of rodents
Gunn
and
suggest
this
trace
additional
metal
in
important
sperm
and
testis.
ACKNOWLEDGMENTS
The authors
wish
to thank
Dr. G. W. Cooper
of
Cornell
University
Medical
College
for photographing
the
preparation
shown
in
Fig.
3 and
Mr.
Walter
Schrepel
of
the
Dept.
of Biochemistry,
Columbia
University,
work
for
performing
was supported
amino
by NIH
Grant
acid
analyses.
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