plasma membrane fraction showed an enrichment in two plasma membrane ..... to note that the membranes from the untreated control cells contained only about.
BIOLOGY
OF REPRODUCTION
1237-1245
32,
(1985)
Isolation and Characterization of Sertoli Cell Plasma Membranes and Associated Plasminogen Activator Activity’ JOANNE
MARZOWSKI,
ROBERT
R.
STEVEN and
GILMONT,
R. SYLVESTER,
MICHAEL
D.
Biochemistry/Biophysics
Program
Washington
Pullman,
GRISWOLD2
State
Washington
University
99164
-4660
ABSTRACT Plasma differential
membranes centrifugation
were
isolated and sucrose
from the density by marker
cultured fractionation.
Sertoli The
cells of 20-day-old distribution and of gradient fractions.
rat testes by purity of subThe plasma
cellular components was determined enzyme analysis membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5’-nucleotidase and ouabain-sensitive Na”/K4’-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5’-nucleotidase and ouabain-sensitive Na+/K+ ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare j35 SI methionineand (3 HI glucosamine-labeled membrane proteins. The incorporation of [35 SI methionine and [3 HI glucosamine was increased in several proteins when the cultured Sertohi cells were treated with retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated
Analysis
of this plasminogen activator as a single 38,0004O,000Mr
primarily
demonstrated form.
that
INTRODUCTION
Sertoli and
cells
play
maintenance
1975;
1978).
One
role of
the creation of a unique ment in which the germinal
in the
control
(Fawcett,
their
recognition
functions
is
et
al.,
1976;
Skinner
and
Wilson Griswold,
of glycoproteins cell membrane shown Zani,
December
Received
‘This Public
August
investigation
Health
Service
Recipient
HD-00263.
1975).
whom
by FSH Wasserman,
synthesis
crude Sertoli has also been
Grant
by United
correspondence
Development should
can
by
other
and
of
provide
insight
these
cells
germinal
be
stimu-
agents
of cell
that
surface
al., 1979). A of the physical
properties which with
by
are
Schuetz,
more and
Sertoli cell into the mecha-
the
interact
with
each
cells.
To learn more of the structures and functions of Sertoli cell membranes, a procedure has been developed to isolate and purify plasma mem-
and
States
branes
from
brane
proteins
cultured
and absence and retinol
Award be
activity
has
partially
purified
ad-
dressed.
1237
Sertoli
cells.
cells
cultured
from
two-dimensional phoresis (PAGE).
HD-17626.
Career
should
they
and
formation”
Haus-
to undergo
when
(Davis
et
cellular
and
known
aggregation
(Bordy understanding
nisms
presence insulin,
was supported
are
culture
“colony
biochemical surface
cells
generally in
(Moscona
FSH and inhibited or inhibit the production
glycoproteins complete
1979;
(Galdieri 1982).
cell
This
by
roles
adhesion
cell
existed
are
essential
Sertoli
in
lated modify
12, 1984. 8, 1984.
of Research To
The
with
1977).
placed
hormone retinol (Fritz
Griswold,
1981).
associated preparations
to be stimulated 1981; Bernard and
Accepted
2
and
man,
and
activator.
enzyme
components
play
homotypic
intratubular environcells, isolated from
follicle-stimulating insulin, and
to
insulin,
of plasrninogen
membrane
believed
serum by the “blood-testis barrier,” undergo various stages of differentiation. The Sertoli cells have been shown to synthesize and secrete a number of proteins in response to hormonal stimulation via (FSH), testosterone,
form
hormone,
the membrane-associated
Plasma
a central
of spermatogenesis
Fritz,
follicle-stimulating
been
of
FSH, been
have
polyacrylamide A plasminogen
The
memin
testosterone, examined
the
by
gel electroactivator (PA)
found
associated
membrane
proteins.
with
the
1238
MARZOWSKI
MATERIALS Cell
AND
METHODS
Culture
Male Sprague-Dawley rats were obtained from the Laboratory Animal Resource Center at Washington State University. Sertohi cells were prepared from 20-day-old rats according to the method of Dorrington and Fritz (1975) as modified by Wilson and Griswold (1979). The Sertohi cells were cultured in serum-free Ham’s F-12 medium (Cibco, Grand Island, NY) or Ham’s F-12 medium supplemented with ovine (o) FSH (0.1 zgJml; NIH-S14, National Pituitary Agency), bovine insulin (5 zg/ml; Sigma Chemical Company, St. Louis, MO), retinol (0.3 tiM; Sigma), and testosterone (0.7 pM; Sigma) (solution referred to hereafter as FIRT). The cells were incubated for 6 days during which the medium was collected, processed, and replenished as described (Wilson and Griswold, 1979). Cell cultures were metabolically labeled with
I’ SI methionine
(400
Ci/mmol;New England Nuclear, Boston, MA) by replacing the medium on the fifth day of culture with methionine-free F-12 and 850 pCi/mI of radioactive methionine for 24 h. Alternately, Sertohi cell cultures were labeled with [ H) glucosamine (50 pCi/mI, 20.2 Ci/mmol; New England Nuclear) in Ham’s F-12 medium for 12 h prior to membrane preparations.
ET AL.
(Emmelot et al., 1974). Ouabain-sensitive Na+/K+ATPase activity was determined by measuring the difference between total ATPase and ouabain-insensitive ATPase activity. The reaction was stopped by the addition of 10% trichloroacetic acid in 5% ascorbic acid. Clucose-6-phosphatase activity was measured by determining the inorganic phosphate released from glucose 6-phosphate (DeDuve et al., 1955; Baginski et al., 1967). The method of Fiske and Subbarow (1975) was used to determine inorganic phosphate released from AMP, ATP, and glucose 6-phosphate in the respective assays. Acid phosphatase activity was measured by the method of McMahon et al. (1977), in which the release of p-nitrophenol from p-nitrophenylphosphate at pH 4.8 is determined. Succinic dehydrogenase activity was determined by
measuring artificial
the succinate-dependent reduction electron acceptor p-iodonitroneotetrazollium
(Pennington, 1961). Protein concentrations were method of Lowry et al. (1951).
violet
Solid-Phase
The
liver
of a 20-day-old
male
Sprague-Dawley
rat
was excised and finely minced in Hank’s balanced salt solution. The minced tissue was then forced through a 1-mm mesh screen. The suspended cells were separated from cellular debris by centrifugation (10,000 X g for 5 mm) through 45% w/v sucrose in phosphate-buffered saline (PBS) and resuspended in PBS. Liver cell plasma membranes were isolated in the same manner as Sertoli cell plasma membranes. Membrane All
out the Linbro,
Preparation solutions
and
fractions
were
kept
on ice through-
procedure. An average of 9 dishes (150 mm; Flow Laboratories, l-lamden, CT) of cultured
Sertoli cells were washed with homogenization buffer (HB) [10 mM Tris(pH 7.4)/i mM KCI/2 mM MgCI2/2 mM phenylmethylsulfonyl fluoridel. Phenylmethylsulfonyl fluoride was omitted from HB when membranes were prepared for plasminogen activator analysis. The cells were then homogenized in a tight-
fitting strokes. for 10
Dounce
homogenizer
The homogenate mm at 0#{176}C to
with
12
was obtain
by
the
was
a modification
iodination on plated
performed of the
procedure
of
Sertoli
Sertohi
described
cells by
cell using
Markwell
and Fox (1978). Sertoli cells grown on 150-mm plastic dishes were washed three times with 0.15 m NaCl/0.05 m Tris (pH 7.4) (TBS) and subsequently overlayed with 10 ml of TBS. Four 22 X 40-mm glass coverslips previously coated with 100 pg 1,3,4,6-tetrachloro-3o,6adiphenylglycouril
(Iodogen, Pierce Chemical Company, were carefully floated on each dish. Approximately 40 pCi of “I (New England Nuclear) were added to each plate. Reaction mixtures were incubated with gentle agitation for 45 mm at room temperature. Cells no longer adhering to culture dishes were decanted and the attached cells were washed three times with TBS. Cell viability after this procedure was estimated to be 85-90% by trypan blue exclusion.
Rockford,
IL)
Cells were harvested, homogenized, and fractionated as previously described with the exception that the crude membrane pellet was washed four times by centrifugation with ice-cold TBS. Radioactivity in the fractions was determined with a Beckman Camma 400.
up-and-down
centrifuged at 3020 X g a crude membrane pellet The pellet was resuspended
and a cytosol supernatant. in 5 ml homogenization buffer, layered onto step gradient of 34%, 40%, and 45% sucrose NaHCO3, and centrifuged at 100,000 X g 0#{176}C.The fractions at the interface of each collected and stored at -80#{176}C. The fraction H9 pelleted onto the 34% sucrose step, H, and H onto 45%, and H, pelleted through sucrose step to the bottom of the centrifuge Enzyme
surface-specific
membranes
Preparation
determined
Radio iodination Sertoli Cells
of Plated
Cell Cell
Liver
of the
a sucrose in 1 mM for 2 h at step were designated
onto
40%,
the tube.
45%
Gel Electrophoresis
Two-Dimensional
Equilibrium
two-dimensional
as
by
done
described
following
modifications:
electrophoresis
O’Farrell
the
was
(1975)
with
the
concentration
of
non-
ionic 0.8%;
detergent (Triton X-100; Sigma) was increased to amphohine (LKB, Gaithersburg, MD) concentrations of 1% pH 3.5-10, 0.5% pH 5-7, and 0.5% pH 2.5-4 were used (Kissinger et al., 1982); and firstdimension gels were run for 10,000 volt-hours. The
second
dimension
acrylamide brane protein
was
run
on
0.85-mm
slab gels. Approximately were loaded onto each
thick
30 gel.
pg
of
10% mem-
Assays
5’-Nucleotidase mining the amount
activity was of phosphate
measured released
by from
deterAMP
Plasminogen
Plasminogen
Activator
activator
Analysis
activity
was
determined
in
SERTOLI
PLASMA
CELL
MEMBRANES
an 11% acrylamide gel containing gelatin and human plasminogen (Sigma) as described by Heussen and Dowdle (1980). Electrophoresis was performed at 4#{176}C at a constant current of 8 mA/gel. Following electrophoresis, the gels were gently agitated at room temperature for 1 h in a solution of 2.5% Triton X100 and then transferred to a solution of 0.1 M glycine
(pH
8.3)
1239
N
N
,,‘
.,‘
‘0
N
00
‘0
‘0
8
en
>5
O1.oo&0. -‘N
‘0
a c)
00 I4
+444+4+4+1
and
incubated at 35#{176}C for 7 h. The gels were fixed and stained for 8 h in a solution of 0.1% amido black in methanol:acetic acid:water (30: 10:60). The gels were destained in methanol:acetic acid:water
O’o’.,l N
“IN
(30:10:60). Fluorography
1.1
+1
*4
*4
-‘
V4
N
00-’
l’n-’
-
Gels
containing
were
samine (Sigma)
SI methionine
(35
or
(‘HI
+4
a
gluco-
as
with 2,5-diphenyloxazole by Bonner and Laskey (1974) sulfoxide was replaced by glacial and Criswold, 1983). Fluorographs
described dimethyl
except that acetic acid (Skinner were obtained on presensitized 1975) Kodak X-OMat AR5 Rochester, NY).
(Laskey film
and
(Eastman
0
N
impregnated
+4
CI)
00 +4
+4
+4
‘0N “I
Mills, Kodak,
00
N
‘ +4
a +4
9 SI
N
6
0
4)
.0
N
‘4
*
N
N
4-4
*4
en“s
N
N
a
a N
RESULTS
0
+4 *4 4-4
0 ‘C
-‘
riOWI*
U “4
Preparation
of Sertoli
Cell
Plasma
o o
Membranes
00
en
,fl
S
-
U a
-
Sertoli
cell
plasma
membranes
were
isolated
+4
+4
N
50
+4
*4
+4
.-‘
Os
s.4 0’
Os
N
from
whole
cell
homogenate
by
a combination a
of
differential
density
centrifugation fractionation.
gradient
brane
and
vesicles
that
formed
Many
sucrose
remained unbroken
‘4‘#5
+4 +4
homogeniza-
in the “cytosol” fraction. cells pelleted through the
a U
CS 0’
Nuclei gradient
*4
-
-
*4
en
-
00
U
+4
0.
00
0’.’
“4
tion and
0
0
-4
‘4
small mem-
during
50env4
N
‘0
‘V
1.0 ‘5
and
were present in the H3 fraction. The preparation and purification membranes from cultured Sertoli monitored with assays of marker
of plasma cells was enzymes for
various
Appropriate
subcellular
enzymes
were
components. chosen
investigations
on
the
(Fleisher
Rosenblum
et
al.,
Na’iK+ATPase
and
as markers phosphatase
for was
basis
and
of
1982).
the used
plasma membrane, as a lysosomal
biochemical
are and
summarized 52% of
and
analyses in the
markers
of
fraction
H0.
of these and H3 The
the
plasma
membrane
two enzymes fractions. specific
activity
was
30% found
in the
N
C
+4
*4
+4
+4
a
a
--
tI’
0 0 .0
‘0
“5
‘0
+1
+4
+4
N
0’.
500
0
+4
44
U ONe4S,-,00 N’0 CS en
>5 N
0
+4
N
2
.5 0
SIC
‘0
-‘en
.9
a
2
U
0 en
“II,
0
a a a
,fl
en en
n
+4 +4 +4 +4 *4
9 0 *4
.0 1/5
U .0 ‘s
501’
0 .5
06
-
U ‘4
in
activity
“cytosol”
‘2
U
0
i’s’s o .
U U
#{176}
.0 0 U)
of
5’-nucleotidase
and (4
ouabain-sensitive H0 showed
0 .0
a
found
of the
V .0
a
‘#1
‘0
0
activity were
Approximately
+4 en
0’
+4
a
chosen
fractions
Na+/K+ATPase
4-4
NNenOO
0
Table 1. Forty-two percent total cellular 5’-nucleotidase
ouabain-sensitive
000
N
U
acid marker,
of subcellular
N
*4
N’ON’
0
glucose-6-phosphatase was used as a marker for endoplasmic reticulum, and succinic dehydrogenase was used as a marker for mitochondria. The
+4
9
1974;
were
U U) U
Ouabain-sensitive
5 ‘-nucleotidase
+4
C”’
0
0
previous
Kervina,
V
en
V U
Na+/K+ATPase 9- to 23-fold
in fraction enrichment of
50
V.
0
V,&
i;
6 >5 N
a
0
-..9
