1. health and fitness
2. disease
3. cancer

BIOLOGY
OF
REPRODUCTION
Cyclic
27,
15 9-168
Adenosine
Monophosphate-Dependent
Human Seminal Plasma:
Origin
and Characteristics
DORIANO
FABBRO,
ALDO
Hormone
Department
AXEL
CAMPANA
and
Laboratory
and
of Gynecology
and
(1982)
and
Andrology
Unit
Protein
of Multiple
Kinases
Forms
JOCHUM,
MARCO
URS
EPPENBERGER’
BALERNA,
Biochemistry-Endocrinology
Research,
Ospedale
University
“La
Cariti
Unit
Clinic
“,
of
Medical
Locarno,
School,
Basle
Switzerland
ABSTRACT
It was recently
cAMP-independent
respective
enzyme
pools,
2)
pendency
can
due
demonstrated
(Majumder,
activity:
be classified
to proteolytic
the protein
kinase
However,
we have
activity
of human
seminal
plasma
was
been able to show that 90 to 95% of the
1) originates
from intra-spermatozoal
cAMP-dependent
protein
kinase
as cAMP-dependent
protein
kinases,
and 3) rapidly
loses its cAMP
deactivities
of seminal
plasma.
that
1978).
These
findings
are supported
by the following
evidence.
seminal
plasma
by 8-N3-l
32P1 cAMP
revealed
the presence
with
molecular
weights
of 52,000,
47,000,
42,000
and
47,000
polypeptides
correspond
to the regulatory
subunits
First, photoaffinity
labeling
of human
of 4 specific
cAMP-binding
activities
37,000,
respectively.
The
52,000
and
R-I and R-II of the 2 cAMP-dependent
isoenzymes,
type
I and type II. The 42,000
and 37,000
molecular
weight
polypeptides
may represent the functional
cAMP-binding
activities
derived
by limited
proteolysis
from
R-l and/or
R-II.
Quantitative
analysis
of the photoaffinity
labeling
of human
seminal
plasma
showed
that
the
37,000
dalton
protein
was the predominant
cAMP-binding
activity.
In contrast,
the cytosol
fraction
(100,000
X g) of spermatozoa
incorporated
only
the 8-azido
cAMP
photolabel
into
R-I
(47,000
Mr)
and
Rll
(52,000
Mr)
Second,
the presence
of cAMP-dependent
holoenzymes
in
human
seminal
plasma
could
also be confirmed
by polyacrylamide
gel electrophoresis
(PAGE)
under
nondenaturating
conditions,
resolving
2 protein
kinase
activities
which
could
not be stimulated
by cAMP
although
they
exhibited
specific
cAMP
binding
and were inhibited
by the rabbit
muscle
inhibitor.
In addition,
a cAMP-binding
peak could
be revealed
corresponding
to the 37,000
and/or
42,000
dalton
polypeptides.
Third,
damaging
the plasma
membrane
of spermatozoa
by
freezing
resulted
in the release
of cAMP-binding
and protein
kinase
activities
into
the seminal
plasma,
which
had the same peculiar
characteristics
of the protein
kinase
activity
found
in the
seminal
plasma
of freshly
liquified
human
semen, i.e. the presence of large quantities
of proteolytic
degradation
products
(37,000
and 42,000
dalton
polypeptides)
and insensitivity
towards
cAMP.
Fourth,
neither
in the seminal
plasma
of patients
affected
by azoospermia
nor in expressed
seminal vesicle
secretion
(EVS)
and expressed
prostate
secretion
(EPS)
were there detectable
amounts
of cAMP-binding
and protein
kinase
activities.
Furthermore,
a positive
correlation
was found
between
the sperm
number
and the amounts
of cAMP-binding
and/or
protein
kinase
activities
in the
seminal
plasma
of freshly
liquified,
randomly
chosen
semen specimens.
Our results
strongly
indicate
that the protein
kinase activity
of human
seminal
plasma
is not an
intrinsic
component
of this fluid,
that it is not produced
by the accessory
male gland
secretions,
and that it may originate
from
the spermatozoa
during
semen
liquefaction.
INTRODUCTION
Cyclic
almost
cess.
nucleotide
every
Accepted
Received
‘Reprint
labor,
Dept.
Schanzenstrasse
systems
aspect
Adenylate
of
February
October
requests:
CH-4031
involved
in
have
in
(Garbers
pro-
reproductive
activities
5, 1982.
13, 1981.
Dr. Urs
Gynakologie,
are
the
cyclase
46,
described
and
tozoa
been
are
Basel,
motility,
the
159
original
to
sperm
In
which
.
1973;
inhibitors
Hoskins
or
and
Carbers
mammals
.
al.,
1978).
spermaexogenous
increase
protein
cAMP-dependent
et
to
respiration
1978;
of
species
addition,
respond
phosphodiesterase
(Garbers
et al.,
The
spermatozoa
(Garbers
Majumder,
Schweiz.
1980).
known
tain
hibit
Universit#{228}ts-Frauenklinik,
Kopf,
well
cAMP
.
Hormon-
mammalian
cyclic
nucleotide
or to exogenous
bolism
1980).
Eppenberger,
several
and
mainmeta-
Kopf,
also
cx-
.
kinase
et
activities
al.,
1972;
160
Recent
investigations
malian
tissues
that
the
found
the
type
I
can
type
al.,
therefore
protein
kinases
proteins
appear
the
for
can
II
suggest
that
and
to
the
their
be
most
as
motility
the
1976;
1978).
One
substrate
likely
candidates
of
and
various
acrosomal
reaction.
It
has
seminal
ties
been
recently
plasma
which
contains
were
1978).
However,
dependency
reported
that
human
protein
kinase
activi-
cAMP-independent
in
and
the
(Majumder,
that
study
origin
of
activity
were
not
established,
whether
the
protein
kinase
from
ity
the
spermatozoa
the
of
reasons
we started
ize
the
ties
of
of
these
enzymes
in
way
for
future
mechanisms
for
an
the
kinase
and
of
be,
activia better
function
sperm
could
these
character-
obtain
biological
elucidation
activ-
For
to
to
clear
derived
intrinsic
plasma.
in order
human
which
male
was
protein
semen
kinase
it is not
an investigation
understanding
ble
was
cAMP-dependent
human
and
seminal
cAMP-
protein
activity
or
human
the
the
in
the
part,
to
pave
of
the
molecular
responsi-
infertility.
MATERIALS
AND
Semen
samples
healthy
donors
were
and
and
Seminal
Plasma
obtained
by masturbation
from
infertile
patients.
After
(30-45
mm
at room
temperature)
the
was separated
from
spermatozoa
by
filtration
through
an isotonic
Percoll
layer (Gorus
and
Pipeleers,
1981).
Semen
(0.5
to 3 ml) was applied
onto 3 ml of a Percoll
solution
buffered
with BiggersWhitten-Whittingham
(BWW)
medium
with a final denseminal
plasma
sity
of
10 mm
cAMP-dependent
regulation
such
activities,
al.,
al.,
of Spermatozoa
liquefaction
of
endogenous
the
from
isoenzymes
et
et
cAMP-dependent
sperm
subunits
(Fleischer
Podesta
Preparation
quantitatively
kinase
1977;
mam-
activities
be
protein
and
et
ET AL.
demonstrated
regulatory
cAMP-dependent
Walter
of
cAMP-binding
fractions
with
a variety
conclusively
affinity
cytosol
correlated
2
have
high
in
on
FABBRO
METHODS
Materials
Bovine
serum
albumin,
benzamidine,
cAMP,
PyroY,
total
histone
type
II A-S,
isobutylmethylxanthine
and
aminophyllmne
were purchased
from
Sigma
(St.
Louis,
MO).
Adenosine
triphosphate
(ATP),
theophylline,
riboflavine
and protamine
sulfate were bought
from
Merck
(Darmstadt,
Germany).
Coomassie
brillant
blue
R-250,
Triton
X-100,
acrylamide
and N,N’-methylenebisacrylamide
were
from
nm
Serva
(Heidelberg,
Germany).
7I32PIATP
(10
Ci/
mmol)
and
I3HIcAMP
(40
Ci/mmol)
were
from
New
England
Nuclear
(Boston,
MA).
8-Azidoadenosine
3’:5’-I32Plmonophosphate
(8-N3-I32PIcAMP)
(60-120
Ci/mmol)
was purchased
from
ICN (Irvine,
CA).
X-ray
film
(Curix
RP1)
was from
Agfa
(Leverkusen,
Germany).
Percoll
and the electrophoresis
calibration
kit
(for
molecular
weight
determination
of
low
molecular
weight
proteins)
were
from
Pharmacia (Uppsala,
Sweden).
Tissue culture
micro
test plates
were
from
Falcon
(Oxnard,
CA).
Millipore
filters
(PHWP
0.45 tm)
were
from
Millipore
(Bedford,
MA),
Instagel
from
Packard
(Warrenville,
IL), and RBYdye
from
Gelman
(Ann
Arbor,
Ml).
All other
chemicals were
of reagent
grade.
1.05 (Rogers
et al., 1980)
and centrifuged
for
at 1000
X g at room
temperature.
The seminal
plasma
remaining
on top
of the Percoll
layer
was
recovered,
centrifuged
at 2500
X g for 10 mm (20#{176}C)
and
assayed
for
cAMP
binding
and protein
kinase
activities
not
later
than
4 h after
sample
liquefaction.
The sperm
pellet
was carefully
resuspended
in 20 mM
Tris-HCI
(pH
7.4) containing
250 mM sucrose,
2 mM
EGTA,
and
10 mM
benzamidine,
and
centrifuged
at
2500
X g for
10 mm
(20#{176}C). The resuspension
was
repeated
and
the washed
spermatozoa
were extracted
for 10 mm at 4#{176}C
with
10 mM Tris-HCI
(pH 7.4), 10
mM
b-mercaptoethanol,
10 mM benzamidine,
and 2
mM
EGTA
containing
0.5%
Triton
X-100
(wlv)
at
approximately
5-20
X 106 spermatozoa/mI.
In a few
experiments
the sperm
suspensions
were additionally
sonicated
in the presence
of 0.5%
Triton
X-100
at
45 W for a total
time period
of 2 mm (in 5 ml plastic
tubes
cooled
in melting
ice) with
a Branson
model
B-12 sonifer
fitted
with a 1/8 in. microtip.
Special
care
was devoted
to avoid
sample
heating
during
sonication.
Following
extraction
and/or
sonication,
the sperm
mixtures
were subsequently
centrifuged
at 2500
X g
for
20 mm at 4#{176}C.Ten
to 20% of the total sperm
cAMP-binding
and
protein
kinase
activities
remained
associated
with
the low speed pellet
(2500
X g). The
resulting
supernate
was further
centrifuged
at 200,000
X g for 30 mm (4#{176}C).The final supernate,
referred
to
as “sperm
cytosol,”
was then
analyzed
for
protein
kinase
and cAMP-binding
activities.
It should
be emphasized
that
both
extraction
procedures
(Triton
X100 extraction
and sonication
in the presence
of the
detergent)
yielded
essentially
similar
protein
kinase
and cAMP-binding
activities.
Prostatic
(EPS)
and seminal vesicle
massages
of men being
treated
for symptomatic
prostatitis
and/or
prostatavesiculitis.
Both
EPS
and EVS were obtained
by the “voiding-bladder”
technique
(VB1
to VB4,
Meares,
1979),
collected
directly
into
1.5 ml conical
tubes
containing
5 M’ of a 50 mM
phenylmethyl
sulfonylfluoride
(PMSF)
solution
in 2propanol
(James,
1978),
mixed and stored
at -20#{176}C.
Before
assay,
both
fluids
were centrifuged
at 5000
X
g for
20 mm
(4#{176}C),and the supernates
analyzed
for
protein
kinase and cAMP-binding
activities.
Protein
32p
Kinase
Assay
Protein
kinase
incorporation
activity
from
was assayed
by
y-(32P1 ATP
into
measuring
protamine
sulfate
(Kuo
and Greengard,
1970).
The
assay
was
carried
out
in a total
incubation
volume
of 300 il
containing
100
g
of protamine
sulfate,
25 mM
sodium
glycerol
phosphate,
10 mM MgCI2,
8 mM NaF,
0.24 mM EGTA,
2 mM theophyllmne,
pH 6.5, and 16.5
MM -y-E32PIATP
(5-lOX
iO
cpm),
in the presence
or
absence
of 3.0 MM cAMP.
The reaction
was started
by
the addition
of 200 M1 of properly
diluted
enzyme
cAMP-DEPENDENT
preparation
was carried
terminated
trichloroacetic
PROTEIN
KINASES
to 100 Ml of the assay mixture.
Incubation
out for 10 mm at 32#{176}
C. The reaction
was
by the addition
of 2 ml of ice-cold
20%
acid (TCA)
containing
0.5% SDS. After
60 mm at 4#{176}C,the precipitate
was filtered
on Millipore filters.
The filters
were washed
3 times with
5 ml
of ice-cold
20% TCA
and dissolved
in 8 ml Instagel
and counted
for radioactivity.
Endogenous
phosphorylating
activity
was determined
in the absence
of exogenous protamine
sulfate
and served
as blank.
The
endogenous
activity
was only slightly
stimulated
by
cAMP
and represented
less than
5% of the activity
measured
in the presence
of protammne
sulfate
and
cAMP.
One unit
of protein
kinase
activity
is defined
as the amount
of enzyme
transferring
1 pmol
of 32P
from
7-132P1 ATP into
protammne
during
1 mm under
assay conditions.
Preparation
Heat-stable
from
rabbit
of Heat-Stable
Protein
Kjnase
protein
kinase
inhibitor
skeletal
muscle
and partially
Inhibitor
OF
treatment
of the muscle
extract
followed
by a
precipitation
(Walsh
et al., 1971).
The inhibitor
was obtained
in 10 mM
Tris-HCI
containing
6 mM
mercaptoethanol,
1 mM
EDTA,
pH 7.4, at a concentration
of 5 mg protein/mI.
This preparation
did not
accept 32P radioactivity
when
tested
as protein
kinase
substrate.
Saturating
concentrations
of the inhibitor
caused
a 99% inhibition
of purified
free catalytic
subunit
activity
of cAMP-dependent
protein
kinase
obtained
from calf ovaries.
Polyacrylamide
Photoaffinity
Labeling
of the Regulatory
of cAMP-dependent
Protein
Kinase
Electrophoresis
(PAGE)
gels
were
prepared
2%
cross-
linked
with
N,N’-methylenebisacrylamide
at total gel
concentrations
of 8%. Stacking
(upper)
gels were
formed
at 3.5%
total
gel concentration
and
crosslinked
with
20% N,N’-methylenebisacrylamide.
Polymerization
and electrophoresis
were performed
as described
by Chrambach
et al. (1976).
Multiphasic
zone
electrophoresis
was carried
out with
the buffer
system
398 (B) at 0#{176}C
consisting
of Tris-chloride-phosphate
buffer
with
an operative
pH of 10.2
(Jovin et al.,
1970).
Gels and cathode
buffer
contained
0.2% Triton
X100 since the recovery
of kinase activity
was found
to
be considerably
improved
in the presence
of the detergent (Salokangas
et al., 1981a,b).
Front
moving
boundaries
were
marked
with
RBY-dye.
The
gels were
sliced
into
1 mm sections
by the use of the Mickle
gel
slicer
(Mickle
Labs.,
Gomshall/Surrey,
U.K.)
and
eluted
overnight
with
300 Ml 01 15 mM Tris-HCI,
pH
7.4, containing
1.6 mM mercaptoethanol
NaCI.
The eluates
were subsequently
tein kinase
and cAMP-binding
activities.
Assay
of cAMP
161
Subunits
The
incorporation
of 8-N3-[32P1
cAMP
into
seminal plasma
(100-200
g
protein)
and sperm
cytosol
(30-50
Mg protein)
was performed
as described
by
Walter
et al. (1977).
The reaction
mixture
(150
MI)
contained
50 mM Mes (pH 6.2),
0.5 mM aminophyl5 mM benzamidine,
10mM
MgCI2,
3 mM EDTA,
1 mM ATP and 0.3 MM 8-N3-(32Pl
cAMP.
Incubations
were
carried
out in the dark,
in tissue
culture
micro
test
plates,
for 240 mm at 4#{176}C.The solutions
were
then
irradiated
with
a Mmneraleight
ultraviolet
lamp
(254 nm) for 10 mm at a distance
of 10 cm. Each sample was finally
mixed
with
50 MI of SDS-Stop
solution
(containing:
30 mM Tris-HCI,
pH 7.6,
50% glycerol,
9% SDS, 3 mM EDTA,
0.02%
bromphenolblue,
0.02%
Pyronin
V and 21 mg/mI
dithioerythrit).
All samples
were
boiled
immediately
for
10 mm
and
electrophoresis
performed
as described.
Polyaciylamide
Quantitation
Polyacrylamide
PLASMA
studies
indicated
that saturation
of binding
was completed
after
180 mm and remained
stable
for at least
20 h. The reaction
was stopped
with
2 ml of ice-cold
10 mM Mes (pH 6.2),
1 mM EDTA,
and 2 mM aminophylline.
Subsequently,
the reaction
mixture
was filtered
by gentle
suction
through
Millipore
filters
presoaked
in the same buffer.
The filters
were washed
3
times
with
3 ml of the above
buffer
and the proteinbound
radioactivity
was measured
by dissolving
the
filters
in 8 m Instagel.
SDS
Gel
SEMINAL
line,
was isolated
purified
by
heat
TCA
Analytical
HUMAN
and 50 mM
assayed
for pro-
Binding
The cAMP-binding
assay was performed
essentially
according
to Walter
et al. (1977).
The reaction
was
carried
out at pH 6.2 in a final volume
of lSOMl
containing
50 mM
2-(N-morpholino)ethanesulfonic
acid
(Mes),
5 mM aminophylline,
2.5 mM EDTA,
100 g of
total
histone fraction
and 81) nM 13H) cAMP.
Binding
of cAMP
was allowed
to proceed
for 240 at 4#{176}C.
Time
of
Gel
8-N3-(
Electrophoresis
and
“P1 cAMP
Incorporation
Electrophoresis
of boiled
samples
was carried
out
in 10% polyacrylamide
slab gels in the presence
of
SDS (Rudolf
and Krueger,
1979).
The gels were run at
25 mA constant
current,
stained
with
Comassie
Blue
R-250,
destained
and dried
exactly
as described
by
Rudolf
and Krueger
(1979).
The dried
gels were then
exposed
to an Agfa
Curix
RPI film with the aid of an
intensifying
screen.
Apparent
molecular
weights
of the
cAMP-binding
proteins
were
estimated
from
a standard
log
molecular
weight
versus
mobility
curve
(Weber
and Osborn,
1969)
using
the calibration
kit
with
the
low
molecular
weight
marker
proteins
of
Pharmacia.
Autoradiographs
of the gels were scanned
in a Pye Unicam
1800
spectrophotometer.
The peak
heights
of the optical
density
tracings
were used as a
measure of 8-N3-(32Pl
cAMP
incorporation
into
individual
bands.
The dried gels were also aligned
with
the
autoradiograms,
the
bands
containing
radioactivity
were cut out and the gel slices counted
directly
with
8
ml Instagel.
In all instances
the peak surfaces
scanned
autoradiographs
were
proportional
total radioactivity
of the corresponding
peak
mated
by liquid
scintillation
counting.
Statistical
Analysis
Statistical
significance
was
test. Correlation
coefficients
ing to Spearman
(1904).
Other
of the
to the
as esti-
Analytical
analyzed
by
were calculated
Student’s
accord-
Methods
Protein
was determined
et al. (1951)
using bovine
by the method
serum
albumin
of Lowry
as standard.
FABBRO
162
Radioactivity
was
(Packard
Insts.).
measured
with
a Tri-Carb
460
C
ET AL.
a
positive
correlation
ferent
from
number
RESULTS
Cyclic
AMP-Binding
Activities
in Human
EPS and EVS
As
shown
in
of
azoospermia.
under
nal
It
our
assay
plasma,
EPS
minimal
and
activity
4 units/mg,
were
per
semi-
assay)
the
cAMP-binding
the
order
of
and
80
contrast
sured
in
chosen
the
sperm
numbers
Donors
and
both
(EPS)
fluids
of
and
30%
106
X
higher
units/mg,
respectively
detection
of
1.
patients:
ml
of
300
ejaculate).
levels
In
and
cases
protein
This
cally
tic
be
subunit
of
of
complex
that
human
inhibited
by
inhibitor
the
heat-
et
(Walsh
inactivates
affinity
90
semi-
al.,
specifi-
cAMP-dependent
a high
the
activ-
1).
protein
activity
in
kinase
activity
muscle
forming
seminal
activity
was the finding
inhibitor
the
kinases
the
protein
inhibitor-cataly-
(Ashby
and
Walsh,
1972).
Labeling
of
cAMP-dependent
8-N3-[32P]
samples
were
donors.
An
was frozen
aliquot
of
immediately
ly
Plasma
and
Kinases
cAMP
Ten semen
spermatozoa/mi)
samples
Seminal
Protein
rest
were
of
analyzed.
500 p1 of each
sample
after
liquefaction
at
fresh
the
seminal
plasma
experiments.
for
the
Figure
frozen
and
semen
and
sperm
label-
that
after
photoaffinity
1A
were
cyto-
subsequent-
plasma
seminal
used
sperm
The
8 days
for
These
were
ejaculates
and
in Methods.
stored
60-100
106
from
healthy
(containing
obtained
shows
photoactivation
of sperm
presence
of
8-N3-[32P1
cAMP,
separation
of the proteins
by
cytosols
in the
followed
by
SDS gel electro-
that
phoresis
2 distinct
activities
in human
the
10
above
found
kinase
1971).
in
sperm
or cAMP-
0.53)
=
(Table
enzyme
could
the
a protein
interest
plasma
rabbit
(r
0.49)
enzyme
± 0.02
the
stable
ing
showed
and
times
we
of
cytosols
kinase
fmoles/mg
addition
of
above
protein
2.5
nal
0.95
particular
95%
=
The
separated
into
sols as described
(P<0.01).
32
and
to
kinase
(r
exhibited
-20#{176}C. The
randomly
significantly
(about
cAMP-binding
32
similar
these
aver-
mea-
1)
activities
than
limit).
the
and
per
were
activities
activities
of
exhibited
which
only
were
(donors
detectable
However,
ratio
with
cAMP-
(Table
plasma
2-140
patients
cAMP-binding
TABLE
fluids.
findings
activities
samples
minimal
plasma
Sperm
3 patients
kinase
these
both
seminal
activities
the
seminal
Photoaffinity
results
prostatic
protein
to
of
semen
samples.
dif-
significantly
between
fmoles/
expressed
(EVS)
for
amounts
the
of
that
pg
activities.
In
age
emphasized
Similar
vesicle
analyzed
binding
by
respectively.
when
seminal
affected
protein
in
the
in
(100-200
EVS
were
obtained
be
levels
mg
and
should
conditions
enzyme
were
patients
5
and
be found
could
the
detectable
and
cAMP-binding
no
activities
plasma
seminal
1,
protein
plasma
ity
exists,
(P<0.O1),
activities
Of
Table
kinase
protein
and
binding
and Protein
Kinase
Seminal
Plasma,
0
and
autoradiography,
seminal
plasma,
prostatic
and
seminal
bands
vesicle
Protein
Protein
kinase activity
(units/mg
protein)
Source
(5)b
Azoospermia
Patients
and
EVS(3)
EPS(3)
aThe
donors
protein
defined
assayed
<4
12.7
<4
<4
(32)
kinase
activity
ratio
±
kinase
2.5’
reflects
0.95
the
degree
of dissociation
as the protein
kinase
activity
assayed
in the absence
in the presence
of saturating
amounts
of cAMP
as defined
bNumbers
in parentheses
indicate
specimens
analyzed.
cMean
±
SEM.
activity
ratioa
(-cAMP/i-cAMP)
of
±
0.02
of cAMP-dependent
cAMP
binding
(pmol/mg
protein)
<80
193
<80
<80
±
58
holoenzymes
cAMP
divided
by the protein
by Soderling
et al. (1974).
kinase
and
activity
is
cAMP-DEPENDENT
PROTEIN
roi
KINASES
OF
SEMINAL
HUMAN
PLASMA
163
SHOWINGTIL PHOTOACTIVATED INCORPORATiON
tto
2-’
SEMINAL PtASlt’
‘p
A.
+
67
43
30
20
FIG.
1. Autoradiograms
the
showing
incorporation
photoactivated
of
8-N3-132P1
cAMP
into
human
sperm
cytosols
and
seminal
plasma.
The
photoactivated
incorporation
of 8-N3-I32Pl
cAMP
was performed
under
standard
assay conditions
in the absence
(-)
or presence (+) of 20MM cAMP.
Seminal
plasma
and sperm
cytosols
were prepared
as described
in Methods.
A) represents
sperm
cytosol,
B) seminal
plasma
of fresh and C) seminal
plasma
of frozen
ejaculates.
=
Mr
of
52,000
the
8-azido
that
70.5%
and
label.
[32P]
cAMP
labeled
26.4%
± 3.4%
associated
with
sperm
cAMP
with
a Mr
tively.
band,
±
band
bands
was
cAMP
The
(Fig.
by
molecular
weights
(47,000
and
52,000)
correspond
II
regulatory
subunits
and
type
dependent
bands
of
labeled
cAMP
active
kinase
protein
porates
specifically
regulatory
subunits
holoenzymes
from
et
Rangel-Aldao
and
a!.,
1977;
Taylor,
1980).
of
the
the
cAMP-
the
photo-
It
cAMP
of
et
tissues
al.,
1979;
has
incor-
the
R-I
and
2 cAMP-dependent
a variety
I
the
R-ll
(Walter
Potter
more
sperm
within
and
frozen
semen
the
reduced
2A
corporation
of
closely
the
(20
protein
seminal
samples
seminal
in
the
sperm
frozen
case
the
while
the
of
that
2B).
the
in
a
of
incorpora-
to sperm
reduced
into
increase
to
protein
and
was even
seminal
fluids
derived
2C).
(Fig.
42,000
band
a slight
decrease
also
was
cross-linked
samples
samples
in-
R-l
significant
label
Fig.
R-l (47,000)
to be signifi-
This
cAMP
of
plasma
cAMP-binding
(37,000)
the
bands
as compared
and
amount
plasma
microof
is shown
to note
by
the
in
pronounced
apparent
4
8-N3-[32P1
paralleled
(P<0.01)
the smallest
latter
specifically
distribution
(P<0.01)
(Figs.
bespecies
cAMP
the 8-azido
label into the
seminal
plasma
was found
of
cytosols
the
was
cold
these
and
distributed
binding
of
of
lB
protein
quantitative
cytosols
or
(Figs.
evenly
excess
pro-
plasma
ejaculates
The
an
label
cantly
type
holoenzymes.
into
of
The
of
predomicytosols
to
that
molar).
seminal
cAMP-binding
above.
by
tion
2
was
4
labeling
the
frozen
label
inhibited
fresh
to
2. It was of interest
ex-
sperm
8-N3-[32P]
analogue
be
6).
the
the
demonstrated
been
recently
of
4
could
or
the
azido
these
micromolar
2, 4,
nantly
it
the
photoaffinity
applied
fresh
described
bands
to
since
a 20
1, lanes
some
respec-
binding
specific
1C),
tween
8-azido
37,000,
=
while
In
the
protein
cAMP
inhibited
of
2A).
was
either
found
same
the
cedure
8-N3-
was
(Fig.
Mr
and
the
SEM)
2 additional
8-N3-[32P1
completely
cess
47,000
42,000
=
The
protein
the
(mean
incorporated
into
showed
of
52,000
When
incorporated
analysis
± SEM)
cytosols
(<5%)
47,000
=
(mean
± 2.5%
only
cases
Mr
Quantitative
became
of
the
of
more
from
In
the
more
8-azido
ET AL.
FABBRO
164
QUANTITATIVE
ANAI.YSIS
PROTEIN
INTO THE VARIOUS
CYTOSOLS
OF 8_N3_(2PJcAl1P
AND SEMINAL
INCORPORATION
BANDSOF HUMANSPERM
PLASMA
100
a)
‘V
0
0
0
C)
0
75
U
C-,’
Co
50
0
C
0
(I,
25
A
B
C
FIG.
2. Quantitative
analysis
of 8-N3-( 32P1 cAMP
incorporation
into
the various
protein
bands
of human
sperm
cytosols
and seminal
plasma.
The autoradiographs
of sperm
cytosol
A), seminal
plasma
from fresh B), and
seminal
plasma
of frozen
ejaculates
C) of the 10 donors
were analyzed
for quantitative
8-N3-I 32Pl cAMP
incorporation
into
R-I (a), R-lI
(0)
and the proteolytic
products
Mr = 42,000
(.) and Mr
37,000
(0) as described
in
Methods.
Results
are expressed
as % of 8-N3-I “P1 cAMP
incorporation
into the various
protein
bands (means
±
SD) of the respective
semen
fractions.
label
present
noticed.
A
in
as seen
label,
with
obtained
by
nal
plasma
during
shown).
In
that recent
work
their
(Potter
and
Effect
Protein
Freezing
any
the
samples
h
at
semi-
37#{176}C (data
can
smaller
functional
Taylor,
polypeptides
1980;
Corbin
cAMP-binding
Distribution
human
addition
of
semen
of
cryopreservative
activities
(Table
from
2).
ponding
sperm
similar
to
of
1978).
by the
activity
agents
re-
of
fresh
measure
cAMP-
and
correswere
However,
for
the
seminal
of seminal
sperm
plasma
and
ejaculates
semen.
between
(activity
the
distribu-
plasma
plasma
cytosols)
and
divided
was
signifi-
higher
(P<0.01)
in the ejaculates
stored
for 8 days at -20#{176}C(Table
2). A similar
distribution
with-
a
activities
spermatozoa
donors
frozen
ratio,
domain
cantly
frozen
of
cAMP-
seminal
kinase
plasma
of
the
cAMP-binding
into
protein
(seminal
those
of
and
sperm
total
cytosol)
distribution
tion
release
kinase
the
The
to note
R-l and
and
10
70-80%
protein
activities
Semen
of
the
binding
which
et al.,
in
not
be proteolytically
cAMP-binding
suIted
dependent
2C),
(Fig.
prepared
it
subunits
be
8-azido
is of interest
has shown
that both
of Freezing
on
Kinase
Activity
in Human
could
of
freshly
10
yielding
retain
subunit
frozen
storing
context
regulatory
degraded,
out
this
R-II
distribution
was
R-II
the
similar
ratio
binding
(Table
evidence
that
kinase
holoenzymes
was
2).
the
obtained
This
for
finding
cAMP-dependent
were
released
the
cAMP-
gives
strong
protein
during
the
cAMP-DEPENDENT
freezing/thawing
seminal
An
cycle
plasma.
intriguing
PROTEIN
from
result
the
was
KINASES
gametes
that
the
into
OF
overnight
taining
protein
in the seminal
plasma
fresh
or frozen
samples
by cAMP,
though
there
kinase
was
inhibition
analyzed
kinase
a 90-95%
activities
muscle
inhibitor.
protein
found
kinase
to
protein
in
be
the
presence
In
contrast
activity
of
kinase
activity
to
ratio
(mean
of
of
this
the
system
experimental
presence
of
protein
is
might
have been responsible
of the
protein
kinases
catalytic
subunits,
the
dialyzed
10
against
mM
Even
the
TM
protein
For
dependent.
analyze
the
seminal
nondenaturating
Seminal
Gel
Plasma
Aliquots
or
TABLE
However,
itself
formed
Human
seminal
by
PAGE
human
of
were
protein
free
and
is not
(Salokangas
et a!.,
and
2 peaks
bind-
of protein
2
peaks
detected
kinase
of
(peak
activities
poorly
kinase
of
A
B.
(Fig.
cAMP-binding
cAMP-
respective
could
protein
ex-
3).
30-50%
activity
protein
the
the
About
per-
amounts
activities
demonstrate
of
A
cAMP.
the
binding
of
on
were
saturating
enzyme
character
and
assays
and
peak
by
inhibitor,
both
cAMP
of
stimulated
muscle
of
findings
A
sub-
kinases
with
protein
cAMP-binding
of
be
holothe
total
as a
recovered
(Fig.
3,fBP),
devoid
of any protein
kinase
activity
representing
small
37,000
and/or
42,000
polypeptide(s)
detected
by photoaffinity
labeling
(see Fig.
C). Only slight
quantitative
differences
could
fro-
nal
50-fold
between
the
electropherograms
from
prepared
plasma
either
of
fresh
the
1B,
be
semior
fro-
zen ejaculates.
cAMP-binding
activities
between
seminal
and sperm
plasma
in fresh
semen.a
and sperm
Source
(10)
(10)
plasma
specific
found
Activity
Fresh ejaculate
Frozen
ejaculate
hibited
such
catalytic
enzyme
were
presence
rabbit
activity
enzymes
under
either
diluted
kinase
the
the
the
These
of
from
if
in
total
buffer
3). The recovery
of enzyme
activities
were
in the order
the
total
activities
applied
were
dependent
render
gel
the
protein
B
kinase
containing
Ejaculates
plasma
ejaculates
2. Distribution
aSeminal
of
plasma
cAMPwe decided
to
Electrophoresis
from
of
fresh
and frozen
ionic
seminal
reasons
plasma
The
high
7.4,
Fig.
8%
Under
free
coinciding
of
cAMP
the
activity,
peak
1970).
gel slicing,
activity,
or
pH
into
was not
aliquots
at
protein
after
B in
conprotein
multiphasic
the
Determining
activities
and
conditions.
Polyacrylamide
zen
of
these
kinase
migrate
PAGE.
and
prolonged
to
70-80%
for the dissociation
into
regulatory
and
seminal
plasma
was
buffer,
able
that
6 mM b-mercaptoethanol.
dialysis
(24 h) did not
Tris-HCI
cAMP-dependent
cAMP-binding
a
dialyzed
subsequently
gels
al.,
et
of
and
by definition
enzyme).
To exclude
the possibility
strength
(Corbin
et a!., 1975)
(Jovin
cAMP-binding
plasma
may
contain
the cAMP-dependent
protein
kinase
holoenzymes
in the dissociation
form
(the
catalytic
subunit
of cAMP-
and
were
the
conditions
kinase
seminal
dependent
protein
kinase
cyclic
nucleotide-independent
using
398
1981a,b).
were insensitive
to cAMP
indicates
that
human
saline
of seminal
plasma
on polyacrylamide
unit
ing
165
ratio
(-cAMP/+cAMP)
procedure.
Dialyzed
concentration
was
a
this
by
p1)
gel
rabbit
result
activity
(200
(-cAMP/+cAMP)
± SEM).
The
RMI-inhibitable
kinase
activities
which
stimulation,
strongly
the
PLASMA
against
Tris
magnesium-buffer
50% sucrose
(see above).
The
affected
protein
sperm
cytosols
and exhibited
cAMP-dependent
of 0.18
± 0.02
large
quantities
of the
SEMINAL
phosphate-buffered
with
kinase
activities
found
prepared
from
either
could
not be stimulated
always
HUMAN
was
separated
of seminal
cytosol
plasma
(total)
Seminal
sperm
plasma
cytosol
activity/
activity
cAMP-
Protein
cAMP-
binding
kinase
binding
Protein
kinase
(pmol/ml)
(units/mI)
(pmol/mI)
(units/mi)
18.1
22.1
31.5
24.8
1.1
11.9
1.1
10.2
±
±
from
of 3.0 MM cAMP
and cAMP-binding
were
1 ml ejaculate.
All values
are expressed
ratio”
are explained
in the text.
3.8
2.8
spermatozoa
performed
as means
and
±
±
3.5
3.5
sperm
as described
± SEM
(n
cytosol.
=
Protein
±
±
kinase
0.8
3.5
activity
in Methods.
Total
activities
were
10). The terms
“total
activities”
in the
±
±
0.4
1.3
presence
calculated
for
and “activity
FABBRO
166
AL.
ET
PAGE OF SEMINAL PLASMA FROM NORMAL HUMAN EJACULATES
16
20
a)
‘a
‘a
12
15
C)
I-)
2
C-
C)
‘V
=
I.’
0
10
0
U
C
CIN
C,
5
‘C
2
GEL SLICE
of seminal
plasma
from
normal
human
ejaculates.
Aliquots
(200 Ml) of dialyzed
seminal
plasma
were applied
on PAGE
as described
in the text. The gels were frozen,
sliced
and elution
was performed
as described
elsewhere,
cAMP
binding
(o-o)
and kinase
assays with
(A_A)
or without
(.-.)
cAMP
and in the
presence
of RMI (a_a)
were performed
as described
in Methods.
FIG.
3. PAGE
DISCUSSION
The
that
results
90
found
the
of
the
first
are
the
semen
supported
intrasperma-
tic
liquefaction.
unit
by
the
there
plasma
the
EVS
between
a
positive
sperm
semen
both
Third,
frozen
proteins
seminal
or
semen
cAMP-binding
their
and
quantitative
the
1978)
according
that
of
spermatozoa
plasma
presence
the
from
demonstrated
that
proteins
are
but
identical,
are
the
that
different.
type
binding
II
it
pure
per
sub-
R2C2
unit
1974;
dissociates
2 C
Corbin
into
sub-
its
equation:
+
R2
(cAMP)4
Taylor,
1978).
not
since
sperm
ence
of
with
et
that
range
of
1978;
only
were
by the
prepared
a competitive
protype
I
cAMP35,000Potter
R-I
and
and
proteolytic
be labeled
benzamidine,
of
functional
1975,
respective
cytosols
that
subunits
a Mr
al.,
surprising
but not
their
were found
to
established
generate
proteins
is
is well
regulatory
isozymes
(Corbin
It
or
of
43,000
cytofresh
samples,
distributions
of
cAMP-
(sperm
derived
and
number.
of
cataly-
overall
holoenzyme
et a!.,
following
4 cAMP
+
Furthermore,
teolysis
chosen
the
sperm
labeling
of
activities
randomly
upon
cAMP
and
the
to
an
The
Rubin
of
dimer
2 identical
with
R2C2.
of
1974;
iso-
found
amounts
kinase
of
indicating
depends
photoaffinity
binding
sol)
al.,
2
subunit
and
(C)
of
types
kinases
contain
and
was
protein
plasma
specimens
azoospermia
cAMP,
of
Lincoln,
units
seminal
II
a regulatory
molecules
et
R2C2
number
seminal
by
type
monomers
4
and
cAMPin
correlation
and/or
activities
detectable
activities
affected
EPS.
cAMP-binding
the
neither
kinase
and
Second,
in
was
nor protein
of patients
and
composition
(Beavo
First,
I
subunit
binds
follow-
evidence.
binding
type
dissimilar
subunits:
(R2),
which
binds
activity
is cAMP-sensitive
from
protein
cAMP-dependent
zyme
evidence
kinase
plasma
during
conclusions
good
protein
originates
compartment
Such
ing
The
the
seminal
probably
tozoal
in
95%
to
in
and
provide
R-Il,
products,
8-azido
in
cAMP,
the
inhibitor
presof
cAMP-DEPENDENT
serine
proteases
and
hence
PROTEIN
also
bin et a!., 1978;
Stambaugh
Zahler
and Polakoski,
1977).
tion
of
the
azido
flect
the
and
subsequently
dent
isoenzymes
label
relative
of
In
to
contrast
always
poration
R-I
lowed
by
an
reduced
and
indicates
that
al.
the
this
(1976)
found
concentrations
protein
almost
would
modulate
present
in
Fourth,
one
has
damage
the
an
assumption
ity
behaved
the
with
by
specificity
In
patterns
could
be
prepared
The
cAMP-binding
of
although
activities,
between
either
increased
frozen
samples
age
of
products
and/or
might
the
acrosomal
in
be
release
enzymes
after
to
for
One
of the
protein
nal
plasma
enzyme
plasma
and
Ashby,
R-II
fluids
of
prolonged
stor-
quantities
cAMP
ished
be
activity
resolved
peaks,
conditions
by
the
and
heat-stable
A
of
could
human
on
PAGE
B,
3).
but
be
rabbit
was that
the
and
(Fig.
cAMP-independent
binding
findings
of
of
the
in
originate
native
holo-
al.,
1977,
et
of
reasonably
explained
can
or
and
RC)
Huang,
give
of
damage
sperm
resulting
protein
also
render
in
membrane
the
B
the
holoen-
Therefore,
that
ejaculates
plasma
and
cAMP-independent
evidence
the
A
considering
1975).
strong
peaks
release
these
liquefaction
at
37#{176}C alter
of
of
human
cAMP-
kinases.
Both
possessed
completely
muscle
semiinto
under
non-
activities
specific
abolinhibitor.
us with
EPS and
EVS.
REFERENCES
freezing/thawing
interesting
activity
could
denaturating
were
most
kinase
composition
which
(Salokangas
proteolysis
supplying
ejaculates.
cycle.
the
a Mr
This research
was supported
in part by the Swiss
National
Science
Foundation,
Grant
Nos. 3.886-0.79
and
3.557-0.79,
and
the F. Hoffmann-La
Roche
Stiftung,
Grant
No. 180. We thank
Dr. G. Colpi,
Dept.
of Urology,
Provincial
Hospital,
Magenta-Milan/Italy,
kinase
R-I
larger
one
that
with
ACKNOWLEDGMENTS
and
seminal
due
of
subunit
tissues,
(R2C2
the
pre-
simi-
seminal
fresh
the
the
demonstrated
exist
cAMP-insensitivity
be
dependent
differences
or
RC
cAMP-
composi-
sub-
plasma
protein
the
frozen
amounts
degradation
sperms,
like
quantitative
detected
from
such
revealed
and
a
activity
a
a subunit
proteolysis
storage
and/or
labeling
plasma
and
(Salokan-
kinase
holoenzymes
that
and
specimens.
seminal
of
The
after
and
seminal
photoaffinity
analysis
PAGE
represent
it was
it
II
type
stimulation,
shown),
of
semen
addition,
PAGE
not
activity
fresh
cAMP
Recently,
(R2C2)
(Huang
activ-
inhibitor,
(data
kinase
from
to
muscle
by
study
effect,
I from
protein
with
mammalian
results
enzyme
might
limited
fact
membranes.
released
respect
rabbit
RC.
100,000
could
freezing,
support
B
fractionSince
the
in this
type
peret
1981a).
cryoprotectors,
semen
The
im-
protein
sieving
enzymes
la,b).
of
the
on PAGE
were
pH (Salokangas
separate
holoenzyme
of
of
a molecular
to
as
various
is
cAMP
liquefaction
by
198
enzyme
possibly
plasma
human
since
inhibition
pared
sperm
of
a!.,
from
pM
it
that
of
during
(-20#{176}C)
protein
anticipate
obtained
possible
zymes
absence
to
results
freezing
to
was
of
et
plasma.
the
mainly
et
because
employed
system
worked
not
167
thus limiting
the “charge
of the PAGE
system.
cAMP-dependent
cAMP-
activity
enzyme
plasma
operative
dependent
human
how
of seminal
at a high
tion
Such
therefore
kinases
formed
was
from
in
understand
seminal
in
strate
range.
analysis
designated
Beck
completely
to
an
the
especially
causes
levels
pM
kinases;
impossible
The
cAMP
the
dissociate
dependent
lar
in
that
the
fol-
protein,
addition,
mentioned
solubility
gas
increased
originates
In
the
are
an
binding
R-I.
that
plasma
by
R-I
be
PLASMA
proved
cAMP-dependent
reguincor-
closely
should
SEMINAL
electrophoretic
plasma
of
protein
of
human
label
and
37,000
latter
proteolysis
seminal
azido
HUMAN
al., 1981a,b),
ation
power”
R-II
degraded
that
followed
in
in
incorporation
closely
and
seminal
reduced
increased
incorporation
limited
present
fact
re-
preparation).
finding,
was
may
cAMP-depen-
proteolytically
of
distribu-
R-I
native
in
The
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the
OF
It
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1972;
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