Itm4Cl - cholesterol in the presence of LI-I. INTRODUCTION. The testis is a complex organ consisting of several distinct cell types including interstitial or. Leydig.
BIOLOGY
OF
REPRODUCTION
23,
Establishment
243-252
(1980)
and Characterization Testicular
of Two
Epithelial
Cell
Distinct
Mouse
Lines
P. MATFIER’
JENNIE
Biology
Department,
University La Jolla,
of
Calijornia,
California
92093
ABSTRACT Two been
cell
lines
cloned
several
epithelial cell
in
lines
One
be
line
isolated
times to
to
(TM4)
have
do
be
from
not
two
been
testis
been
of
tumors
distinct
cell
and
when types
identified
testis
is
a
several distinct or Leydig cells, cells.
mone
complex
much
Each
of
these
progress
has
difficult
part,
to
the
al.,
function
Primary
the
of
problem
while
contain
organ
cultures
of
Steinbergen, Sato
the
each
isolated
percentage
their
Sertoli
of
has
morphology,
cell
origin,
from
types
cells
of
not maintain periods of Steinberger,
function
(Sato
1970). interstitial
Cell
cell
however,
lines are responsiveness
for
long
have
capable and periods
of
but
by either metabolism
LII
Yasumura,
serum (Mather
cell
may
it
lines
their in
1971
a,b;
et
with and
1978),
another
time
of
it
FSII
shows
a
not
or
LII.
of
FSII, Itm4Cl
1966;
Sato
been established of both the
maintain
Shin
some,
interstitial
al.,
but
cells
1968).
There
not
-
et al,, from and
rat all,
of
the
many report
such
(Shin,
however,
are,
equivalent in the media to replace
and growth Bottenstemn
factors et al.,
to
possible
nontumorigenic
retained present
in vivo
from that of recent advances chemically-defined and the ability
hormones Sato, 1978;
was
of
of their describes
in
establish
clonal
testicular
cells
vivo functions. the characteristics
cell that The of
lines,
shown of maindifferentime
MATERIALS
in
Cell
1.2
cultures
of
becco’s 1980.
AND
modified
g/l
of
Ham’s
sodium
cells
maintained
nutrient
Eagle’s
15% (v/v) horse serum (FCS)l . All media and
25
15
mM
units/mI and were
ampicillin),
a 1:1
Dulwith
N-2-hydroxyacid
Mg/mI (US) scra
and
supplemented
-2-cthane-sulfonic
(192
in
mixture
medium
bicarbonate,
(F12/DME), antibiotics ig/ml streptomycin,
243
were
F12
ethylpiperazine-N’
Received March 30, 1979. Requests for reprints should be addressed to Dr. J. P. Mather, The Population Council, The Rockefeller University, 1230 York Avenue, New York, NY 10021.
METHODS
Culture
mixture
29,
and
which
Stock
April
hormone
since
of
lines have cell tumors
a manner different normal cells. With composition of (Waymouth, 1972)
in
one
cell
culture
two
coworkers,
that cultured taining hormone
Accepted
of
are l’hc
few
1975). and
of
1967;
due,
suffer
each
inherent difficulties in interpreting data obtained from transformed cell lines, as these lines can lose hormone responsiveness or respond in
experimental each cell type than
have
characteristics
type
be
lines
steroids.
mouse
under-
cell
more
and
last
may
cultures
frequently do for prolonged
(Steinberger
culture
tiated
in
This
containing
a small
type and functions
into basis
These
2 years. The cells BALB/c nu/nu mice.
horAlthough
a complete of
achieve,
to
individually.
type,
made
1976),
of
distinctive
the difficulty in obtaining in which one can study
systems
the
of
as being
interstitial cells and
responses.
been
et
of
been
consisting
has
and
(Hansson
standing
organ
cell types including Sertoli cells, myoid
requirements
years
injected on
mice.
over
for
purified
INTRODUCTION
germ
culture
ovFSH and an increase in cAMP in the presence line (TM3), believed to be Leydig cells, is not growth-stimulated increase in cAMP in the presence of LII, and a change in the in the presence of LI-I.
The other shows an cholesterol
The
in
metabolism
tentatively
BALB/c
immature
established
form
gonadotropins,
has
to
and
and
said
responses response
growth
been
appearance
can
response,
have
(II EPES),
penicillin.
200
and
serum
2.5% fetal calf obtained from
serum Grand
MATHER
244 Island
Biologicals,
Inc.
The
cells
were
tissue culture dishes in a humidified 95% air:5% CO2 at 37#{176}C.Cells were 5-7 days. Experimental All
on plastic
atmosphere subcultured
were
mixture with (SF-Fl 2/DME). Cells from
carried
the omission Hormones exponentially
out
in
the
inoculum in 60 mm tissue culture dishes required hormones had previously been numbers were obtained by removing the plate with trypsin, neutralizing with an of serum-supplemented stock medium, using a Coulter counter. All experiments ed 2-5 times over a period of 6 months. stimulation experiments were terminated all conditions were subconfiuent. and
same
of the antibiotics were added where growing stock
cultures were removed from the plate with (0.1% w/v). The trypsin was inhibited with volume of soybean trypsin inhibitor (0.1% the cells washed with SF-F12/DME. Cells suspended in SF-F12/DME and plated at the
110 rmones
of every
Procedure
Culture experiments
F12/DME and sera indicated.
grown
trypsin an equal w/v) and were reindicated
to which
the Cell cells from the equal volume and counting were repeatAll hormonewhen cells in added,
containing
1 mM
1-methyl,
was
Chemicals obtained
from
Collaborative
114 Cl-cholesterol (50 mCi/mmol) were obtained from New England Nuclear and were >98% pure as assayed on thin layer chromatography. Samples were counted in a Beckman beta counter (counting efficiency 45% for H and 65% for Metabolism
medium was changed and i HI -progesterone 106 cpm) or 1’ Cl-cholesterol (3.6 X i0 added. Twenty-four hours later, the medium collected and the steroids extracted twice with methylenedichloride (80:20). The extract was dissolved silica gel radioactive
in absolute thin layer steroids,
(2,X cpm) was etherdried,
ethanol, and a portion spotted on plates. After application of nonthe chromatogram was developed
in benzene:acetone (80:20). The plates were dried, dipped in 20% penchloric acid, and heated to visualize standards. The plates were then cut into 5 mm strips and the radioactivity counted in toluene/omnifluor.
Cyclic
AMP
The
two
isolated specific tion
Assays
Cultures were plated in 60 mM dishes in SFF12/DME with the addition of insulin, transferrin and EGF and grown to confluency (4 days). At time 0 the medium was removed and replaced with SF-F12/DME
lines
Lines
testicular
much
of
the
removed. cells
the
medium
or
or
The
testicular
tissue pieces
cells with
were very
fibroblast
growth,
FSH
ng/pl).
(100
to
as
dispersed tubule.
serum-free (