BIOLOGY
OF
REPRODUCTIONNewly
35,Synthesized
179-190
(1986)
Proteins
and Intratubular
ROBERT
in Seminiferous
Compartments
B. SHABANOWITZ4
and
of the Rat Testis’
ABRAHAM
Department
Intertubular
L. KIERSZENBAUM2’3’4
of Anatomy3
and
The
The
Laboratories
for
Reproductive
Biology4
School
of Medicine
of North
Carolina
University
Chapel
Hill,
North
at Chapel
Carolina
Hill
27514
ABSTRACT
used
fluid
Two-dimensional
to characterize
(SNF).
Fluids
gel electrophoresis
newly
synthesized
were
collected
combined
with autoradiography
and Western
blot procedures
have been
proteins
in testicular
intertubular
fluid
(TIF)
and seminiferous
tubular
following
in vivo and in vitro
intratesticular
injection
of
Sf methionine
135
into
control
and hypophysectomized
detected
within TIF and SNF.
Their
labeling
conditions.
While
two major
after
Three
in
in vivo
acidic
TIF
ous
labeling,
proteins,
samples
tubules
these
two proteins
possibly
secreted
collected
and
TIF
Mr
72,000
acidic
monal
deprivation.
after
in vitro
protein,
possibly
Autoradiographs
breakage
and
found
in TIF
can gain
to both
TIF
1w SI methionine
rats
reflecting
of TIF
leakage
during
and SNF have
and
changes
and most
spermatogenesis
and extratesticular
les.
of the
Because
niferous
(TIF),
which
intertubu!ar
blood
vessels),
stream
targeted
spermatogenic
to developing
physiological
whose
major
the
surrounds
components
transports
after
labeling.
of the
intertubular
epithelium
SNF,
showed
caused
many
Results
of this study
origin
and that proteins
seminifer-
intensity
suggest
secreted
of the
by pituitary
protein
spots
that
by
horthat
most
albumin
the Sertoli
cell
molecules
from
cells
and
TIF-borne
the
Sertoli
(Dym
and
Fawcett,
fluid
intratubular
compartments.
Cultured
rat Sertoli
cells
synthesize
and
secrete
proteins
that
are serum
like (Skinner
and Griswold,
1980,
1983;
Wright
et a!., 1981),
follicle-stimulating
hormone
(FSH)-dependent
(DePhilip
and
Kierszen-
and
and
blood
baum,
baum,
by a
1975)
are
tubules
cyclically
1983).
1982)
1982;
of
by
The
and cell-specific
(DePhi!ip
Kissinger
et al., 1982).
the
rat,
the
Sertoli
the spermatogenic
protein
composition
from
the intertubular
rete testis compartments
and Kormano,
1973;
Hinton,
1884.
Dept.
of
217-H,
Chapel
cells
semi-
developing
molecules
spermatogenic
cells is regulated
barrier
(Setche!!
and
Waites,
seminiferous
tubular
components
and
1970).
Serto!i
cells
are
also
likely
to contribute
molecules
to the TIF,
thus
establishing
a reciprocal
link
between
the
seminiferous
intertubular
and
seminiferous
tubules
(mainly
Leydig
cells
Accepted
November
25, 1985.
Received
September
6, 1985.
‘This
study
was supported
by USPHS
grant
2 Reprint
requests:
Dr. Al L. Kierszenbaum,
University
of North
Carolina,
106 Swing
Bldg.
27514
Incubated
135 SI methionine-labeling
in
in the seminiferous
remarkably,
of
sample
collection.
an extratesticular
in vivo
after
in vitro labeling.
were more
abundant
SNF.
organization
of
in samples
collected
45,000
and 35,000),
peritubular
testicular
for
Serto!i
cells.
Access
1w sj methionine-labeled
proteins
were
varied
according
to in vivo and in vitro
and transferrin,
were radioactively
labeled
than
a decrease
requires
the interaction
bioregulatory
molecu-
avascular
epithelium,
of
labeled
72,000,
labeling
showed
INTRODUCTION
Mammalian
of testicular
number
abundance
albumin
were
insignificantly
by Sertoli
cells (Mr=
of hypophysectomized
suggested
cell
and transferrin
access
adult rats. A discrete
presence
and relative
blood
plasma
proteins,
1985).
testicular
and
sized proteins
HD1
Anatomy,
Hill, NC
ment.
179
and KierszenIn seminiferous
cell
is influenced
cycle
(Wright
et al.,
of fluids
collected
(TIF),
intratubular
has been reported
Turner
et a!., 1979;
However,
extratesticular
accumulated
little
is known
(SNF)
and
(Koskimies
Olson
and
about
the
origin
of newly
synthein each testicular
compart-
180
SHABANOWITZ
AND
In this
paper,
we describe
the two-dimensional
po!yacry!amide
gel
electrophoretic
patterns
of
[35 S] methionine-labeled
proteins
collected
from
interand
intraseminiferous
tubular
compartments
after
in vivo and in vitro
labeling.
Results
from these
experiments
were
correlated
with
[35 SI methioninelabeling
experiments
that used incubated
segments
of
seminiferous
and differences
tubules
to determine
possible
in the patterns
of proteins
[35 51 methionine
in
electrophoretic
and
allowed
identification
intraseminiferous
testicular
vivo
and
in
Western
blot
of
proteins
tubular
and
similarities
labeled
with
vitro.
Combined
approaches
have
in interand
compartments
extratesticular
that
are
of
origin.
MATERIALS
AND
KIERSZENBAUM
tomized)
testes
were
At
the
adult
rats
(2
wk
with
and
after
Sf methionine
hypophysectomized
hypophysectomy)
River
Breeding
Labs.,
Wilmington,
tized
with
ether.
A small
portion
exposed
through
a scrotal
incision
length,
and
England
500
pCi
Nuclear,
> 1000
Ci/mM)
was
tunica
albuginea
of
carried
in 100
um
containing
methionine
p1
of
slowly
Eagle’s
10%
the
Animals
essential
concentration
meth).
Scrotal
(by
the
killed
experi6 h for
ether
for collection
of both
of Sharpe
and Cooper,
method
of Turner
et al.,
anesthesia
TIF
(accord1983)
and
1984).
Blood
TIF
and
SNF
were
mixed
1:1 with
lysis
(O’Farrell,
1975)
and used directly
for deterof
trich!oroacetic-acid-precipitable
radioSamples
were
stored
at -20#{176}C for no more
2 wk
prior
to protein
In vitro [35 SI methio
sample
collection. Adult
electrophoretic
nine-labeling
rats (intact
studies.
of
testes
and
or hypophysec-
with
Testes
were
to incubate
containing
[35
the
5] methi-
placed
in organ
for 6 h at 32#{176}C
5% CO2 in air.
period,
TIF
samples
processed
as
and
SNF
described
SiMethionine-labeling
of segments
of isolated
seminiferous
tubules.
Segments
of seminiferous
tubules, free
of intertubular
tissue,
were
collected
by
mechanical
means
in Hanks’
balanced
salt solution
(HBSS)
from
sexually
a stereomicroscope.
were
mature
rats
Approximately
segments
of
comprising
(as judged
seminiferous
all fourteen
by their
(17 X 16 mm)
supplemented
pg/mi)
examined
25 randomly
patterns)
were
rinsed
in
tissue
culture
100
amino
U/mI
containing
200
pCi
h labeling
containing
time
5%
and
was determined.
thionine-labeling
niferous
tubules
acids
and
fresh
wells
of
and
buffer
and
5] methionine.
[35
at 15,600
removed
in lysis
meth
giuta-
(0.1 mM),
streptomycin,
at 32#{176}Cin
CO2
in air,
centrifuged
in
tubules
(2-5
mm
stages
of spermato-
containing
300 pl of EMEM-10%
with
sodium
pyruvate
(1 mM),
mine (4 mM), nonessential
antibiotics
(penicillin,
100
and
transillumination
collected.
The
segments
and placed
in flat-bottom
supernatant
was
were reconstituted
were
allowed
cleared
from
injected
anesthesia;
[35
removed
of
ether
incubation
and
The
ples
this labeling
step,
of blood
was col-
by deep
the
was
mediincisions
of
of
the
most
suitable
for
optimal
labeled
proteins
from
the
were
and testes
removed
ing to the method
than
minimum
normal
Following
and a sample
end
was
beneath
radiolabel
and
After
a 16
atmosphere
by cardiac
puncture
with a heparinized
syringe.
was centrifuged
in Wintrobe
tubes
and plasma
collected.
activity.
just
The
activity
circulated
for 6 h. Preliminary
that
a circulation
period
testis
(data
not shown).
rats were
anesthetized
mination
testis.
of
[35
SI methionine
was
recovery
of radioactively
SNF
injected
each
(New
specific
with
surgical
clips and rats
Injected
[35 SI methionine
the testes
and
ments
indicated
plasma,
buffer
SI methionine
deep
above.
HBSS
(Charles
MA) were anestheof each testis
was
of about
1 cm in
MA;
(EMEM-10%
were
closed
to recover.
lected
Blood
[35
Boston,
and
[35
removed
collected
were
genesis
In vivo labeling
of testes
sample
collection. Intact
by
onine
as described
above.
culture
dishes
and allowed
in a humidified
atmosphere
selected
in length)
METHODS
killed
were
a humidified
the medium
X g for
3 mm.
lyophiized.
Samand radioactivity
A 16-h-period
was used for [35 SI meto allow
isolated
segments
of semito equilibrate
with
the incubation
medium.
Preliminary
experiments
indicated
cant qualitative
differences
between
6 and
no signifi16 h incu-
bation
times
except
for greater
recovery
of secretory
proteins
(data
not shown).
Two-dimensional
polyacrylamide
gel electrophoresis (2D-PAGE).
Aliquots
of radiolabeled
samples
containing
100,000-300,000
cpm were
analyzed
by 2DPAGE
according
to
the
method
of O’Farrell
(1975)
with
minor
modifications.
Isoelectric
focusing
gels
contained
ampholytes
(pH 5-7:
1.6%,
pH 3.5-10:
0.4%,
Serva
Fine
Biochemicals,
Garden
City
Park,
NY).
In some
experiments,
0.4%
of ampholytes
pH
3-5
were
added
to improve
resolution
end of the gel. Samples
were focused
volts
for 16 h followed
by 800 volts
20-mm
incubation
time
in equilibration
second
dimension
gradient
slab
electrophoresis
was
gel.
to
run
Sample
prevent
at the acidic
at 400 constant
for 1 h. After
a
buffer,
the
in a 5-15%
was
not
protein
logarithmic
heated
before
carbamylation.
NEWLY
Molecular
way, NJ)
dimension
weight
(Mr) markers
were run in a separate
of the electrophoretic
determination
tional
experiments,
of
Mr
electric
point
(pI)
(Pharmacia,
lane during
procedure
resolved
standards
were
set of
protein
coordinates
spot more
have
we
same
and
resolved
used
Mr
by 2D-PAGE
and
visualized
after
were
partially
sham,
of all
paper
in a gel
X-OMAT
ter,
for
dryer.
1-2
wk
transferred
1979).
Gels
buffer
containing
carried
for
out
in
1#{189}
h. After
gelatin
for
TBS
the
in distilled
Blue.
dried
according
The
gels
and
dried
on
exposed
Kodak,
Roches-
developed
resolved
in Kodak
to nitro-cellulose
were
soaked
20%
the
paper
overnight
methanol.
same
blots
antirat
were
treated
(TBS),
pH
Malvern,
PA)
at
containing
3%
volts
with
7.4,
reactive
with
sites;
rabbit
(both
from
transferrin
was
at 65
1%
for
45
subseantirat
Cooper
a working
dilution
of
bovine
serum
albumin
After
this incubation,
the blot was
30 mm
with
five
changes.
The
incubated
with
peroxidase-conjugated
washed
washed
in TBS for
blot
was
goat
antirat
IgG (Cooper
Biomedical)
in a sealed
plastic
bag for 1
h at room
temperature.
Immunoreactive
sites were
detected
with
4-ch!oronaphtho!,
and the blot
was
rinsed
in distilled
water.
Blots
containing
immunore-
film
radioactively
labeled
proteins
mm in Amplify,
dried
and
as described
were
exposed
soaked
for
to X-ray
Newly
synthesized
by
intra
proteins
testicular
TIF
with
and
[35
SNF
SI me-
1A,B),
attempt
was
proteins
antibodies
to individual
Samples
onine-!abe!ed
using
generated
which
made
to
a Western
against
human
and
not
a p1 range
[35
proteins,
protein
protein
reported
proteins
et al.,
of
and
in TIF
Seminiferous
with
from
plasma.
4.0-4.4
(Fig.
intratubular
5] methiplasma.
Two
[35
and a p1 of 4.6
protein
had
a
p1 of 4.5
samples
S] methionine
including
(Fig.
1C).
blood
at least three
seen in blood
a Mr “ 34,000
The
other
of Mr34,000
but
with antibodies
raised
and to the unavailabi!(besides
transferrin
and
proteins
of TIF displayed
proteins
not
(Fig.
1B).
was
seen
in blood
1A,
arrowhead).
samples
in
vivo
albumin
However,
and
of testis
labeled
displayed
the
a Mr=7
large
A
various
2,000
acidic
number
of
spots,
some of them
corresponding
to proteins
previously
as stage-specific,
intracellular
from
the
seminiferous
tubules
(DePhilip
1982),
suggested
that a release
of intracellular
proteins
occurred
one
may
conclude
proteins
detected
testes
and
low
labeled
during
SNF
collection.
Therefore,
that
nearly
all major,
labeled
in plasma
and
TIF,
including
transferrin,
concentration
are
in
either
SNF
absent
collected
or at
from
in vivo.
It was of interest
whether
hypophysectomy
would
yield
changes
in the electrophoretic
patterns
of blood
plasma
and
TIF
proteins
labeled
with
[35 SI methionine.
Patterns
of plasma
proteins
collected
from hypophysectomized
rats injected
intratesticularly
with
[35
SI methionine
from
These
control
included
showed
several
the
in
those
Mr34,000
Figure
found
cell
changes
leakage
occurred
protein
in
control
suggested
during
rats
that
the
2A).
of
indicated
1A. Protein
patterns
in SNF
rather
than
plasma
proteins
observed
Figs. 2B and C). This finding
tial
significant
rats
(compare
Figs.,
1A and
a reduction
in the labeling
intensity
and
an arrowhead
resembled
in
labeling
(Figs.
An
plasma
reaction
of rat antigens
human
plasma
proteins
rat-specific
antibodies
transferrin
above.
RESULTS
detected
positive
against
ity of
albumin
extremely
Incubation
was performed
2 h at room
temperature.
collected
from
were
essentially
plasma
proteins.
We were
unable
to assign
specific
names
to any blood
plasma
protein,
with the exception
of albumin
and transferrin.
This was due to a lack of
plasma
2D-PAGE
transfer
buffer
proteins.
of the
with
Mrr45,000
(Towbin
et
in transfer
Protein
methanol
transfer,
by
proteins
of these
proteins
had
(Fig.
1B,
arrowheads).
manufacturer’s
of proteins
2D-PAGE
of the proteins
in hF
derived
Of major
significance
was the
of albumin,
transferrmn
and
plasma
approach
protein
Proteins
and 1% normal
goat serum.
in sealed
plastic
bags for
acted,
10-15
blot
plasma
some
albumin)
(Amer-
gel was
to the
in Tris-buffered-saline
Biomedical,
1:100
in
rinsed
(Eastman
at 40#{176}C
to block
nonspecific
quently,
the blots
were
incubated
or rabbit
for
blood
major
identify
of a
paper,
Mr
30 mm,
mm
serum
position
this
in vivo.
Patterns
determined
by
to
other
by a
in Amplify
at 70#{176}C
and
blotting.
were
a!.,
were
The
film
GBX X-ray
developer
recommendations.
Western
IL)
AR
suggested
that
most
from
blood
plasma.
[355]
methionine-labeling
autoradiograms.
soaked
Heights,
In addiand iso-
similar
181
TESTIS
thio nine
TIF
and
determined
Coomassie
destained,
to Kodak
NY)
with
IN RAT
Piscatasecond
to allow
together
calculated
gels
staining
Arlington
filter
p1 were
in several
Two-dimensional-PAGE
water
PROTEINS
proteins.
Coomassie
with
corresponding
that
defined
the
accurately.
Throughout
an average
protein
the
protein
spots.
of known
Mr
with
[35 SI methionine-labeled
Blue-stained
gels were
matched
autoradiograms,
SYNTHESIZED
by
of TIF
blood
(compare
a substan-
collection
of
182
SHABANOWITZ
AND
KIERSZENBAUM
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n
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PROTEINS
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184
SHABANOWITZ
AND
KIERSZENBAUM
TIF
S
#{149}‘
aib
4.
-
Tf
72
--
.
(b
0
Op.
S.
/
-
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#{149}
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45
0
In
.
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#{149}
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000
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A
B
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#{149}
.5-
a-rat
FIG.
3.
[“SI
Methionine-labeled
not found
in SNF
that
may
be Sertoli
rat transferrin
(Tf,
these
labeled
prominent
proteins
samples.
Dotted
lines
cell-specific.
Western
C). The Tf immunoblot
two
samples
from
with
[35 SI methionine
acidic
in TIF
(A)
following
in vitro
with
Mr=3
rats
two
5,000
and
45,000
increased
their
intensity
in TIF as compared
to SNF
samples
(Fig.
2B).
In addition,
TIF samples
displayed
an upward-projecting
acidic
protein
of
Mr3
5,000
(Fig.
2B, arrowhead)
that
was also conspicuous
labeled
samples
Mr28,000,
also
in
samples.
When
tomized
in TIF
in vitro
samples
of hypophysectomized
(Fig.
5A, arrowhead).
In
consistently
p1 range
Figs.
1C, 4A
comparing
and control
displayed
two
5.7-5.9
(circle
and 5B), not
SNF
rats
of intact
rats.
C
Upward
arrowbeads
indicate
TIF-specific
proteins
indicate
negative
image
of albumin
(aib) and transferrin
(Tf).
Boxed proteins indicate
secretory
products
blots
of TIF after
labeling
with
[“SI
methionine
and probing
with rabbit
antirat
serum
(B) and rabbit
antishows
a major
immunoreactive
product
of Mr_76,OOO
as well as several
breakdown
products
of lower
Mr.
hypophysectomized
in vivo. However,
proteins
labeling
Tf
samples
labeled
turn,
proteins
in Fig.
detected
from
in vivo,
rats
SNF
of
2C and
in TIF
hypophyseca substantial
decrease
was
noted
in the
labeling
intensity
of a
Mr72,000
protein
(compare
Figs. 1C and 2C).
Newly
synthesized
proteins
in
TIF
and
SNF
detected
by intratesticular
labeling
with
[35
Sf methionine
in vitro.
A large
number
of [35 SI methioninelabeled
proteins
were
detected
in TIF
following
intratesticular
vitro
and
(Fig.
3A).
range
Mr3
injection
of
incubation
of whole
Three
acidic
proteins
4.4-4.6),
Mr=45,000
5,000
(p1 range
4.4-4.7)
In particular,
displayed
proteins
considerable
This
behavior
containing
sialic
[35 5] methionine
in
testis
for 6 h at 32#{176}C
with Mr=72,000
(p1
(pI range
4.0-4.5)
and
were intensely
labeled.
with
Mr=72,000
and 35,000
charge
microheterogeneity.
is characteristic
acid or other
of
charged
glycoproteins
sugar moieties
NEWLY
(Pearson
and
Anderson,
1983).
SYNTHESIZED
In
PROTEINS
addition,
TIP
strings
S] methionine-labe!ed
samples
of
Mr immunoreactive
TIF
that
contrasted
(Fig.
3A) in
(Mr72,000,
weakly
However,
were also present
When
radioactive
labeled
serum
range
(data
of
not
detected
5.3-5.7
these
shown).
a major
as well
proteins
were
Rabbit
Mr-76,600
as both
lar
transferrin
protein
horizontal
with
and
labeled
with
blot
[35
after
analysis
The
(Fig.
3C).
of TIP labeled
TIF
proteins
that
pair
detected
were
of
only
Mr=28,000
after
SI methionine
intratesticu(Fig.
1C)
labeling
in vitro (Fig. 4A).
samples
of SNF were
probed
by
using
al proteins-including
vertical
samples
to three
35,000)
SNF.
proteins
labeling
Western
p1
and
in
SNF-specific
radioactively
antirat
proteins
with
regard
45,000
were transferred
to nitrocellulose
paper
and immunoreacted
with
a rabbit
antirat
serum,
a large number
of
immunoreactive
proteins
were
detected
(Fig.
3B).
none
185
TESTIS
of lower
pattern
in vitro
4A).
[35
RAT
Samples
of SNF from testes
labeled
with
[35 5] methionine
in vitro
(Fig. 4A) yielded
an electrophoretic
samples
labeled
in vitro
showed
several
proteins
with
Mr less than
43,000
and p1 greater
than 4.7 (Fig. 3A,
arrowheads)
not observed
in SNF samples
(compare
with Fig.
When
IN
in vivo
rabbit
antirat
serum,
albumin-showed
sever-
immunoreac-
SNF
aib
Tf
alb
72
-Il
/
I
/
/
.
45
0.
0
.-1
0
r.
I
-i
#{149}
C
p
a-rat
B
serum
A
Tf
/
aib
/
#{149}
0
-0
that
FIG.
are
position
blots
of
munoblot
reaction
4. Proteins
SNF-specific.
in SNF
Boxed
labeled
protein
with
13S SI Methionine
indicates
a secretory
following
in vitro
product
(Mr=72,000)
a-
rat
labeling
that
of
may
C
Tf
intact
adult
rats (A).
be Sertoli
cell-specific.
The
circle
Dotted
(alb)
of Sertoli
cell-specific
proteins
Mr=45,000
and 35,000.
Dotted
lines indicate
negative
images
for albumin
SNF
after
labeling
with
[35
SI methionine
in vitro
and probing
with rabbit
antirat
serum
(B) and rabbit
antirat
shows
a major
immunoreactive
product
of Mr=76,000
and several
apparently
low Mr breakdown
products.
in C may be due to nonspecific
Tf adsorption.
indicates
a set of proteins
indicate
the expected
boxes
and transferrin
transferrin
(Tf,
The positive
(TJ).
Western
C). The
Tf im-
albumin
(aib)
186
SHABANOWITZ
tivity
(Fig.
antirat
4B).
When
transferrin,
the
Mr76,600
protein
samples
of TIF
rin-immunoreactive
lower
SNF
Mr variants
was
a strong
labeled
(F.ig.
4C).
(Pig.
collected
(Pigs.
enough
and
of TIP or SNF
[35
SI methionine
labeling
to be detected
after
from
As
observed
in
2B).
the finding
radioactively
after
3A
in vivo
image
that
occurs
Protein
patterns
from
from
hypophysectomized
larly
with
[35 SI methionine
similar
in vitro
and 4A)
little
within
labelcom-
gels
labeled
that
was
(Fig.
in vitro
TIF
(Fig.
3A) was weakly
labeled
rats
labeled
in vivo
in
and
(Pigs.
2B
and
the
the
in vitro
(Figs
heavily
of
vivo
in
testis.
nor transferrin
of TIP or SNF
labeled
in
labeled
that
changes
in
protein
reflected
rats
samples
5A).
This
finding
expression
of the
effect
of hormonal
were
from
radioactively
hypophysec-
5A,B).
accumulated
in TIP
and
SNF were
compared
with
those
detected
in the medium
of incubated
seminiferous
tubules
as an approach
for detecting
proteins
originating
in cellular
components
of the seminiferous
of
tubule.
We found
the
patterns
of incubated
and
hypophysectomized
observed
in animals
labeled
in vitro.
Figure
that
samples
of TIF from
hypophysectolabeled
in vitro display
a Mr=3 5,000
acidic
thionine:
Mr45,000
following
differences
seminiferous
tubules
rats labeled
with
(1)
the
loss
of two
from hypophysectomized
acidic
rats
in protein
of control
[35 5] meproteins
as compared
of
I
0
Tf
#{149}
S
#{149}
1
/
LI
in
rats
Newly
synthesized
proteins
in segments
of isolated
seminiferous tubules incubated with [35 Sf methio nine.
The
patterns
of
[35 SI methionine-labeled
proteins
autoradiograms.
t
labeled
tomized
samples
of TIP
and SNF
rats
labeled
intratesticuin vivo were
correlated
TIF-H
found
protein
deprivation
on the
Neither
albumin
labeled
in samples
not
and
high
or no synthesis
the testis.
that
A Mr72,000
suggested
Mrz72,000
(com-
Blue-stained
in the
to
hypophysectomized
the TIF-control
rats
both
hypophysectomized
that neither
labeled
in
conditions
on Coomassie
a negative
findings
indicate
or transferrin
with those
5A shows
mized
rats
at
Figs.
lB and C). However,
although
in
autoradiograms,
both
albumin
were
present
in
concentrations
to generate
These
albumin
detected
these
samples
ing with
pare
with
detected
transferrin
protein
rabbit
was
4C).
was
were
to their
with
KIERSZENBAUM
in vitro (Fig. 3C), the transferproduct
was also associated
with
Of particular
interest
albumin
nor transferrin
pared
probed
reaction
AND
.
.,
.
-
Tf
-alb
F
S
I
I
S
g
0’s’
SI
[135
-
II
S
I3)
a
A
B
FIG.
5. [ SI Methionine-labeled
proteins
in TIF-H
(A) and SNF-H
(B) of hypophysectomized
rats labeled
in vitro.
The upward
arrowhead
indicates
a heavily
labeled
protein
seen in TIF (compare
with Fig. 2B, which
illustrates
TIF of hypophysectomized
rats labeled
in vivo).
Boxed
proteins
indicate
secretory
products
that
may
be Sertoli
cell-specific.
Spots
within
some
boxes
are overlapping
proteins.
The circle
indicates
SNF-specific
proteins.
Dotted
lines indicate
negative
images
for albumin
(alb) and transferrin
(TJ).
NEWLY
SYNTHESIZED
PROTEINS
IN
RAT
187
TESTIS
ST-H
ST-C
Tf
-
-
/
-.
4
IS.
45
-
___
4
j_A’’___
_______
*
C
--
I _i:i,,,
,,
.__.C
I
S
A
C
7
a-rat
B
Tf
FIG.
tomized
6. Proteins
labeled
with
35
SI Methionine
(B, ST-H)
rats. Upward
brackets
(identified
ward bracket
(identified
as A)
indicates
proteins
cell-specific.
Traces
of transferrin
(Tf)
are indicated
nfl (TI).
to control
reduction
rats
(labeled
A
in labeling
intensity
of Mr56,40058,500
the Mr=7
compared
Figs.
of three
(labeled
B in Figs.
6A,C)
and
labeling
intensity
of a string
of proteins
of Mr=37,900
p1 range
5.0-5.2,
from
hypophysectomized
rats
compared
to control
rats (labeled
C in Figs. 6A,C).
as
TIP
in hypophysectomized
rats,
and
(3) the
(2) the
proteins
as
in
sized
protein
control
6A,C),
acidic
rats
increase
The
acidic
which
2,000
to
in
synthesize
and secreted
by isolated
seminiferous
tubules
of control
(A, ST-C)
and hypophysecas B and C) indicate
differences
in protein
labeling
intensities
between
these
two samples.
Downmissing
after
hypophysectomy.
Boxed
proteins
indicate
secretory
products
that
may
be Sertoli
in A. B illustrates
an immunoblot
of control
seminiferous
tubules
using
rabbit
antirat
transfer-
most
prominent
proteins
proteins
of Mr=72,000,
appear
to be identical
proteins
and
of similar
Mr
in various
samples
by Western
proteins
blot
did
not
analysis,
cross-react
[35
in
serum
labeled
transferrin
(data
not
shown).
However,
transferrin
cross-reacted
with
serum
(Fig. 6B). This finding
that some transferrin
vitro
incubation
of
was newly
isolated
synthesized
segments
of
tubules.
DISCUSSION
of
SNP
When
probed
thionine-labeled
suggested
during
seminiferous
in both samples
were
45,000
and
35,000,
to three
newly
synthe-
seen
rabbit
antirat
radioactivity
rabbit
antirat
5] mewith
We
resolve
have
used
2D-PAGE
newly
synthesized
and autoradiography
proteins
labeled
to
with
[35 5] methionine
present
in seminiferous
inter
and
intratubular
fluid- compartments
of the testes
of adult
control
and hypophysectomized
rats. In control
rats,
serum
proteins
appear
to provide
the major
contribu-
tion
to
TIP
as
demonstrated
by
[35
SI methionine-
188
SHABANOWITZ
labeling
experiments
Sharpe
(1981)
human
albumin
less
than
21/2
Therefore,
the
[35
ing
performed
testis,
Setchell
and
showed
that
the clearance
of [125 I]
from
rat testes
takes
place
slightly
hours
after
intratesticular
injection.
in vivo
intratesticular
injection
of
SI methionine
used
newly
synthesized
the
in vivo.
AND
enter
in our experiments
proteins
should
the
systemic
6-h labeling
period
tissues
for incorporation
and
for
rapidly
circulation
detectclear
within
the
become
available
to other
into proteins.
Newly
synthe-
sized
proteins
produced
in tissues
other
than
the
testis
and released
into
the blood
could
then
reach
the testis
by the systemic
circulation.
Everett
& Simmons
(1958)
reported
that the testis
had
the
most
intravenous
other
tissue.
rapid
gain
injection
Setchell
in
[131
II albumin
(3 16% at 1 h), greater
& Wallace
(1972)
also
after
than any
showed
a
KIERSZENBAUM
1985).
The
albumin
sistent
in the SNF,
as reported
with
these
studies.
The
[35
tured
of
no
onine-labeling
period
used
thus
could
TIF
periments,
and
radioactivity-labeled
contention
albumin
synthesized
in our
[35
in vivo
contribute
proteins.
SI methi-
labeling
ex-
to the pool of
Supporting
the
is
the
finding
of radioactively-labeled
and transferrin,
two proteins
known
to be
in the liver,
in both
blood
plasma
and
lar
organization
testes,
et
and
and
which
of
the
intertubular
is essentially
al., 1973).
When
correlates
are
port/secretory
place
them
in
or
the
in the
space
established
between
in vitro
[35
5] methionine-labeled
SNF samples,
it can be observed
and transferrin
are
sequent
to in vivo
two labeled
plasma
space
a lymphatic
rat
vivo
patterns
both
of TIP
albumin
5] methionine-labeled
labeling.
The occurrence
proteins
in SNF suggests
only subof these
a trans-
that
[35
synthesis/secretory
seminiferous
tubular
pathway
lumen.
to
Al-
though
a functional
blood-testis
barrier
exists
in the
testis (Setchell
& Waites,
1975), iodine-labeled
albumin
has been
shown
to pass, albeit
slowly,
into the tubular
lumen
(Setchell
& Wallace,
1971).
The
immuno-
histochemical
localization
of albumin
has also shown
low concentrations
of albumin
in the lumen
(Christensen et al., 1985),
and albumin
has been
shown
to be
present
in SNF in small
quantities
after collection
by
micropuncture
(Koskimies
and
and
evaluated
Kormano,
by
1973;
gel
Olson
electrophoresis
and
Hinton,
and
of rabbit
C] amino
transferrin
the
liver.
rat
is available
on
little
is known
reported
that
synthesize
Griswold,
and
1980,
cul-
secrete
1982)
and monkey
incubated
acids. In addition,
the
receptors
51 methionine
[35
absence
in
and
testicular
source.
in TIF
for 24presence
spermatogenic
in vitro?
cells
has
Steinberger
et al.,
why is no labeled
testicular
labeling
The
explanation
of albumin
was previously
cell is known
to synthesize
in SNF is dependent
upon
its presence
for
stated:
since
this protein,
a blood
plasma
The presence
of radioactively-labeled
albumin
or SNF is strictly
dependent
on whether
plas-
ma albumin
is labeled
testicular
labeling
with
viding
an explanation
cult. One
toli cells
(Fawcett
in
[14
thionine-labeling
TIF after intratesticular
injection
of [35 SI methionine
in vivo.
In addition,
the similarity
in protein
patterns
of blood
plasma
and TIF is supported
by the particu-
is the
been
the
labeled
in this paper,
is conmost
likely
source
of
been reported
(Holmes
et al., 1983;
1984).
However,
a question
arises:
albumin
or transferrin
seen
after
to
6-h
(Skinner
ous tubules
48 h with
returned
the
It has
from
of
have transferrin
receptors
(Holmes
et al., 1983;
Sylvester
and Griswold,
1984).
Thorbecke
et al. (1973)
have reported
the synthesis
of transferrin
by seminifer-
with
during
cells
transferrin
amount
information
in the testis,
transferrin.
Sertoli
a small
albumin
considerable
of albumin
concerning
turnover
of iodine-labeled
albumin
in the
lymph.
Therefore,
it is likely
that proteins
at extragonadal
sites
could
easily
be
testis
of
S] methionine-labeled
Although
the behavior
very
rapid
testicular
synthesized
the
presence
transferrin
as one would
expect
only after
[35 SI methionine
in vivo. Proof the insignificant
[35 SI me-
of transferrin
possible
are not
as they
in SNF
interpretation
heavily
engaged
are in culture.
is rather
is that,
in the
In fact,
diffi-
in vivo,
synthesis
there
Serof
is much
less synthesis
of transferrin
incubated
seminiferous
tubules
(Fig.
6A) than
in cultured
Sertoli
cells (authors’
unpublished
data).
A possible
gene activation
in cultured
Sertoli
cells correlates
with
a recent
report
that
these
cells acquire
a 13-adrenergic
response
during
culture
(Kierszenbaum
Hypophysectomy
causes
of
a Mr=72,000
cally
in
5B). This
Mr45,000
SNF
acidic
samples
protein,
and
et al.,
a decrease
protein,
seen
testes
labeled
of
along
35,000,
1985).
in the
with
may
most
synthesis
dramati-
in vitro
(Pig.
two other
proteins
of
be the same
proteins
that
several
laboratories,
including
ours,
have found
to be synthesized
and
secreted
by cultured
Sertoli cells (Wright
et al., 1981:
proteins
S4, S8 and S7;
DePhilip
designation:
and
Kierszenbaum,
S70, S45 and
proteins
4, 5 and 6).
It is not
surprising
secretory
binding
proteins
in
protein,
another
1982,
and
S35; Kissinger
to
find
Sertoli
TIP
samples
since
Sertoli
cell-specific
unpublished
et al., 1982:
cell-specific
androgensecretory
NEWLY
protein
has
(Gunsalus
been
reported
et al.,
1980).
to
be
SYNTHESIZED
secreted
Secretory
PROTEINS
into
proteins
that
TIF
origi-
nate in Sertoli
cells may play important
intragonadal
roles
in the regulation
of spermatogenesis.
Indeed,
DePhilip
et al. (1982)
and Wright
et al. (1983)
have
shown
changes
in
protein
synthetic
activities
of seminiferous
stages
of the rat spermatogenic
shown
that
decreases
(1)
its
Sertoli
cell-specific
a condition
and (2)
which
comigrates
5,000
protein
but
intensifies
its labeling
sectomized
rats.
It is likely that
synthesized
by
components
compartment,
1983).
in TIF
include
a
by
(Sharpe
1985),
the
et al.,
influence
upon
1981;
which
during
the
collection.
similar
proteins
that
were
also
paper,
function
Fritz,
immediately
increased
lysis
or weakening
possibility
of cell
as did
collection
SNF
of
are
samples
whole
or SNF collected
paper.
As reported
overnight
Turner
time
of the
tubular
such
[35
that
might
include
more
when
we isolated
seminiferous
tubules,
patterns
to those
of TIF. Furthermore,
of whole
seminiferous
significant
transferrin
from
tubular
5] methionine-labeling
shown
proteins
that
can
labeling
in
the
vivo,
role
they
of these
may
be
We
the
space
cells
in
vivo
have
vectorial
as compared
intertubular
Testicular
and that
according
conditions.
that
study.
a discrete
be observed
compartments,
abundance
varies
suggesting
lumen.
this
of SNF colmay not be
to
in
toward
may
be
of liver oriby Sertoli
cells.
proteins
important
is yet to be
as intrago-
regulators.
Bergh
A
1982.
Local
differences
in Leydig
cell morphology
in the
adult
rat testis:
evidence
for a local
control
of Leydig
cells
by
adjacent
seminiferous
tubules.
Int J Androl
5:325-30
Christensen
KA, Komorowski
TE, Wilson
B, Ma S-F, Stevens
RW, 1985.
The
distribution
of serum
albumin
in the rat testis
studied
by
electron
microscope
immunocytochemistry
on ultrathin
frozen
sections.
Endocrinology
116:1983-96
DePhiip
RM, Kierszenbaum
AL, 1982.
Hormonal
regulation
of protein
synthesis,
secretion,
and phosphorylation
in cultured
rat Sertoli
cells. Proc NatI Acad
Sci USA 79:6551-55
DePhiip
RM, Tres LL, Kerszenbaum
AL, 1982.
Stage-specific
protein
synthesis
during
rat spermatogenesis.
Exp Cell Res 142:489-94
M, Fawcett
DW, 1970.
The blood
testis
barrier
in the rat and the
physiological
compartmentalization
of the
seminiferous
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