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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 (