Vol.
154,
August
No. 15,
3,
1988
BIOCHEMICAL
IETECTION
CELLULAR SOMAT
RETINOL-BINDING
IC
CELLS
OF
lstituto
di
THE
lstologia
ed
lstituto
di
SC i enze Chi
Received
cDNA
as
a
June
28,
clone to
these
specific
mRNA.
In
effective
in
spermatogenesi cells
mammals
Sertoli
because
cells
are
(FSH)
Napol
i,
di
di
Roma
Medicina
e
Naples
Protein gene
in ccl
gene
and two
when
compared
cells
In the
this
higher Sertol
we of
by
with
the
paper
the
the
hormones
testosterone. times
used of
synthesis
modulated
FSH
was
somatic
Is). in
is
(CRBP)
the
involved
as
takes
maturation
(1,2).
from
the many
lular
Moreover, concentration i
ccl
of
Is.
Michela 14,
cells the
the
such their and
$1.50
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
1174
of
action their
fol
this
but
process.
Sertoli hormone
these
cells
secretory
Istologia
only
epithelium
act
necessary
di
are
not
I icle-stimulating on
which
tubules
spermatogenesis
of as
where in
the
seminioferous
microenvironment
Gal dieri, Istituto ROMA 00161, Italy
tubules
compartment
regulation
hormones
the
seminiferous
adluminal
of
hormonal
exert
the
somatic
metabolism
create
Scarpa
the
These
which
to: A.
in
architecture
mediate
ccl
place
completion
the
testosterone
to
basal
normal
for
contributes
Copyright All rights
Facolta
approximately
occurs.
‘target’
their
0006-291X/88
I I
tubular
the
levels
the
the
they
Via
peri
such
an
generate
regulating
Correspondence Generale,
THE
Rams
the
actively
cells
development
and
IN
Inc.
they
also
and
s,
revealed
of
because
of
are
Sertoli
separate
responsible
Universita
Retinol-Binding
i
cells
spermatogenesis
cell
,
di
expression
mRNA
cells
Qerm
enra’
RNA
V.Colantuoni$
Generale,
Sapi
Cellular
the (Sertol
that
peritubular
TUBULES
and
Universith
a,
for
study
of CRBP steady-state 0 1988 Academic press,
SEMINIFEROUS
1174-1181
1988
tubules
demonstrate
RAT
Biochimiche,
rurgi
coding
probe
seminiferous
In
COMMUNICATIONS
MESSENGER
Embriologia
‘La
A
RESEARCH
PROTEIN
R.Faraonio$
M.Galdieri”:,
$
BIOPHYSICAL
Pages
OF
‘:
AND
1988
directly, iv i ty
for
ed
Embriologia
germ
which cell
Vol.
154,
No.
Retinol
is
(3,4,5)
and
for
the
the
cellular
the
cells
3,
1988
essential in
OS t eron
e ,
transferrin
gene
intracellular believed of stochemical
present
in
binding peritubular
cells
cDNA
been press
to
togenesi
MATER
I
ccl
CRBP
and
al
(19).
a
which
vitamin the
(14). Sertol
the
The
i
ccl
Is
amount
CRBP
has
high
level
been of
i
by
of
tubules a
soluble
retinol
both
in
the
Sertol
CRBP
has
been
recently
CRBP
mRNA.
This
clone
this
paper
to
study
and
in
the
specific in
the
somatic
ccl
Is
and
of
the
acting
on
isolated
(18)
the
seminiferous
regulation
of
the
has the
its
maintainance
of
50
to
Sertoli
After
MEM)
seteded
into
humidified
culturing male
the
ml
medium.
Falcon
Petri
smal
I
fragments
con
ical
tube
addition
outgrew
by at
of
Equal at from
the
1175
and
2
and min,
then minimal
Eagle’s aminoacids,
to
amounts
initial
of
n iferous
pipetting for
non-essential
air
method
semi
(Gibco
containing 95%
seminiferous the
gentle 75xg
solution) dishes
of to the
medium
defined
atmosphere cells
explants according
treatments,
penicillin-streptomycin 1
I
rats
enzymatic
ly
with
smal
Wistar
the
chemical
U/ml
aggregates a
by
graduated a
medium
glutamine,
culture
in to
a in
in
the
recently
cells)
old
dispersed
resuspended
kept
of in
hormones
prepared
in
ly
in
the
20-22-day
wlere
centrifugecl
usual
to (CRBP),
seminiferous
rat
peritubular
by
were et
essential
binds
the
METHODS
from
Dorrington tubules
of
protein
reported
gene
preparation Is
thelium
with
and
AND
ccl
Sertoli
proteins
5.
IALS
Sertoli
culture
the
exerted
sperma
and
transcription
and
human
experiments
(Sertoli
expression
of
the
of
epitheiium
to
cross-hybridize
in
i on
of protein
specific
retinal
More
detected
corresponding
usecl
the
radioimmunoassay been
into
(17).
clone proved
and
the
targets
composition
insulin of
evidenced
of
be
mannose
ide
FSH,
effects
cells
has
of
retinol-binding
been
to
ipid-dependent
cells
(15,16).
specific
protein
ar
has
different a
)
biological
methods
appear
secretion
target
spermatogenesis
glycopept
with the
Iul
the
normal
calcium-phosphol
the
ccl
protein
by
the
(9,10,11
COMMUNICATIONS
incorporation
changes
Is In
mediate
the
quantitatec
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the
this
immunohi
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the
influences
(12,13).
to
fact,
interacting
A Sertol
Sertoli
their
Moreover,
carrier,
presence
ex
inhibits
vitamin by
and
in
RESEARCH
of
tubules
(6))
(8).
BIOPHYSICAL
maintainance
increases,
and
sinthesized
A
the
glycoconjugates (7)
AND
seminiferous It
activity
is
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the
vitamin.
kinase t est
BIOCHEMICAL
a
ratio
of
this
the
culture
5% explants
CO2.
4 of
0.1
were
suspension medium After and
2-3 formed
mM
ml and days
of a
Vol.
154,
No.
3, 1988
monolayer
overlaid
Hormonal
by
i cells used
Peri tubular ______---------------Peri tubul
ccl
(20,
material
detached
rous
Usually
The
Northern
blot
described
Dot
blots
held
in
was
and
the
on
denaturing
were
ug
of
was
As
of
5
the
isolated
seminife-
The
pellet
was
min.
The
pelletted
with
10%
fetal
of
within
using
15
or
the
3
method
RNA
95%
discarded cells calf
air
days
of
of
serum
and
5%
C02.
culture.
20
Chirgwin
et
was
established
preparation gel
ug
ccl
of
RNA
in
al
(22). by
(23).
each
lane
was
performed
in
fi Im
dot
10
RNA
to
were
so
samples
quantitative LKB
of with of
scanning
and
considered
to
total
denatured
trascan and
only
in
laser
I I
Sertoli
ug
added a
abundance
ul
of 10
that
were
an
filters was
relative
by
amounts 0.5
lulose RNA
ug)
The
using
of
nitrocel Yeast
filter.
calculated
range
to
and
dot. the
various a
blot
of
analysis
densito-
peritubular
the
values
subsequent
that
RNA
to as results
higly
in
the
calcula-
CRBP
contain is
synthesized
a
good
to
extract
Five
and
ten
the
cDNA
preparations two
amount
them.
determined
by
densitometric
demonstrate
for
in the
Sertoli
cells.
corresponding in
these
1176
used
RNA
coding
tested
and
of
similar
Since
it
that has
(17)
our
the in of
the been
the
fig.1
scanning time
were for
and
intensity
first
protein cells.
of
RNA
to
The
RNA
the
ug
clone
were of
total
cross-hybridization
of
expressed
they
to
with the
used
gene.
give
obtained
These is
to
different
preparations
autoradiograms.
CRBP
were
hybridized
proved
proportional
RNA
the and
previously
was
rats
prepuberal
expression
hybridization
that
(5
each
was
RNA (24).
abundance.
Five
previously
sample to
from
counterpart.
CRBP
RNA
sensitivity
prepared the
the
apparatus
DISCUSSION
analyze
CRBP,
total
Id
in
cRBP
mRNA
Is
human
applying
ifo
standards
relative
AND
various
min. for
obtained
autoradiographs
used of
for
for
for
were range
I
2
preparation
the
atmosphere
each
application
internal
RNAs
spotted
signa
of
formaldehyde-agarose
each
the
of
shows
the
previously
culture
supplemented
of
included
coding
RESULTS
rat
300x9
humidified
by
of
RNA
mRNA
meter.
and
for
at
prepared
man
before
i
40x9
was
prepared
densitometry
Sertol
procedures
cell
treatment
MEM
yield
template
formaldehyde
ticns
a
using
quantities
Ii near
and of
analysis
RNA
analysis
a
different
cell
culture
(20).
on
the
of
concentrations
to
Sertoli
at
in
37°C
and
electrophoresis
cells.
legends.
collagenase
monolayer
integrity
as
the
confluent
RNA isolation __---------------Total cellular
The
figure
COMMUNICATIONS
beginning
according
centrifuged
at a
germ the
culture.
during
resuspended
cultured
RESEARCH
associated at
of
the
centrifuged
were
and
3
isolated
by
supernatant
obtained
day
Briefly,
was
the
of started
in
were 21).
tubules
and
at
BIOPHYSICAL
preparation
cells
employed
amount
indicated
I
AND
usually
u ti I ized are
ar
small
were
were
hormones
establish
a
treatments
Sertol
10
BIOCHEMICAL
gene shown
findings
the the
Vol.
154,
No.
Fig.1.
3,
Dot
Five
ug
were
question 5,
exposed
to ture
was these
extracted
single
mRNA
species
mRNA
precursors.
whereas
The is
was
The
asn
increase
of
approximately
in
combination
used
SD
of
CRBP
mRNA
cells of
CRBP as
a
were
mRNA relative
day
of
of
used
in in
the the
detected
in
density
of
cells.
1177
the
hormonal amount
recognizes weight of
lanes
observed 4).
the
to 2
when
Fig.2
B
CRBP
Sertoli
compared A,
was
and
molecular
times
lane
were
blot
to
two
each
effective
contamination
experiments different
the
to
the
of
probe
higher
in
to
Northern
(fig.2
A,
were
RNAs
testosterone
about
Is
values
cDNA
A
active
ccl
total
CRBP
times
(fig.2
the
by
separately three
B
be
dose
culture
and
i
single
of
due
FSH
and
combination
A
pairs.
mRNA
detected
maintained
range
probably
CRBP
hormones
the
base
in
ture.
The
of
of
cul
to
Sertol
or
bands.
administration
induction
goal
analyzed
700
known
this
cells,
observed
the
!SertoIi
expressed
sometimes
when
-+
amount
about
the
A
P
experiment.
the
third
32
a
cRBP.
hormones,
in
treated
of
this
preparato
human
alone
of
of
for
To
legend),
and
species
in
cell
hybridized
coding
gene.
the
COMMUNICATIONS
different
and
added
on
two
whether
this
figure
mRNA
from
was
RESEARCH
RNA.
filter
beginning
(see
densitometry
were
utilizing
testosterone the
Is
used
and
by
an
hormones average
preparations
control
caused
control
fragment
Routinely
from
quantitateld
cDNA
affect
used
cells.
lulose
could
BIOPHYSICAL
extracted
nitrocel
addressed
from
ccl
RNA
we
FSH
media
hormone
cells
RNA
AND
Sertoli
total on
two
spermatogenesi
one
of
spotted
the
next
on
of
nick-translated
indicate
cul
analysis
ten
labelled
BIOCHEMICAL
blot
and
t ions
The
1988
and
3)
the
two
shows
the
performed cultural ly detected
conditions. treated
Sertoli in
the
the
cells control
Vol.
154,
No.
Cyclic ex
a
BIOCHEMICAL
1988
AMP
was
on.
Sertoli
pressi
at
3,
also
tested cells
concentration
analyzed t ive
experiment.
CRBP
mRNA
data
about
the
conditions
expression
used,
3
FSH of
it
seems
FSH
RNA
representa-
the
level
from
control
are
able
least
in
of
the
cells. to
the
testosterone
and
extracted
one
in
At
and
RNA of
testosterone gene.
CRBP AMP
the
results
the
the
dibutyryl-cyclic
induction to
COMMUNICATIONS
stimulate
and the
an
CRBP
that
to
shows
and
the
could
days
compared
that
it
three
caused
times
RESEARCH
exposed
for
treatment
show
whether
Figure
three
presented
positively
mM
blot. The
BIOPHYSICAL
therefore,
0.1
Northern
of
check
were,
of
by
to
AND
The regulate
experimental
have
an
additive
*
02
1
Fig.2.
A
2
Nothern
stimulated ug
of
i
ccl
total
was
coding
for
were
hybridized
testosterone The
3=
testosterone
Fig
.3.
arrows.
RNAs
Sertoli
of were
cells. fig.2
3
control
P
labelled
and
4
hormonal
cells;
2=
( RNAs
0.5
uM
used
ly
of
the
l=
control
4=
dibutyrilxyclic from
FSH
and
AMP
on
control
1178
and
0.125
Iulose
ovine
of
2=
and
0.125 4=
respectively). markers
tmRNA FSH
detected
treated
is in
the
cells;
cells.
levels.
dbcAMP the
S-16, cells;
treated
mRNA
The fragment
weight
testosterone CRBP
FSH
ug/ml
CRBP
1.5%
paper.
treated
molecular
cells;
are
legend.
(NIH uM)
levels
(2)
conditions
on
cDNA
(0.5
as
cells. cells;
nitrocel
FSH
testosterone cells
size-fractionated
nick-translated
Mean+SD -
experimental
lane, onto
B
extracted The
from
each
Sertoli treated
Effect
3=
ribosomal
treated
in
control
treated
of by
ly
RNA
tranferred 32
a
l=
I 5;
FSH
migration
hormonal
in
ccl and
indicated
The
cRBP.
loaded and
to
treated
ug/ml)
of
2.
Is.
gel
human
1
5
analysis
RNA
formaldehyde-agarose filter
4
blot
Sertol
Twenty
3
same
(0.1 as
mM) those
treated described
(1)
Vol.
154,
effect,
No.
3,
in
line
secretory
proteins
In
to
order
present
in
blot
with
compare cell
cells
are
where
the
CRBP
(17).
for
revealed
tlnat
and
2).
three
i,
cell
types
also
in
cells
the
Sertol
i
ccl
I
days
gene
To
CRBP to
a
the
cells
specific
confluent
monolayer.
at RNA
and cells
it
analysis lanes
1
appears
times
that
more
in
than
these
two
influence
could low
and
(fig.4,
protein
time
Sertoli
blot
mRNA mRNA,
plated
than materials
two
culture
tubules
RNA
the
that
cells.
Sertoli
CRBP
of
were
Northern
CRBP,
(see
approximately
if
of
seminiferous
prepare
the
abundance
evaluate
gene, get
ative
with
peritubular
confluency.
the
compared
higher
to
synthesize
this
the
isolated
reached
of
rel
from
concentrations were
levels
express
(17).
at
they
level
of
utilized
u nt i I
Sertoli
high
component
animals
the
of ten
mRNA
COMMUNICATIONS
for
extracted
cells
these
paralleling
expression
literature
contain
detected
same days
cells
Sertol
RESEARCH
the
RNAs
somatic
tubular
Comparing
peritubular
for
the
to on
been peri
CRBP
known
other
has
The
cultured
BIOPHYSICAL
in
of
performed the
from
level
types
was
methods)
reported
the
other
analysis
Is
data
AND
(9,10,12).
These
ccl
BIOCHEMICAL
1988
density
extracted
the
and from
these
cultured cells
-28s
N-18
1
Fig The
.4.
CRBP RNA5
experimental
mRNA were
levels
in
extracted conditions
are
2
Sertoli from the
s
and
peritubular
Sertol
i
same
as
1179
(1) those
and
cells. peritubular described
(2) in
fig.2
cells. legend.
The
Vol.
154,
was cul
No.
A
in
not
conclusion
som
at i c
of
the
and
intensity
did
not
modify
BIOPHYSICAL
RESEARCH
compared
to
was
obtained,
the
level
COMMUNICATIONS
that
from
short-time
indicating of
CRBP
that mRNA
the
synthesis
shown).
from
the
the
RNA is
shown
the
mirror by
spermatogenetic
coding
protein
some
we
tubules,
gene
the
regulated
here
seminiferous
express
steady-state gene
experiments
of
actively
the
same
culture
components
Is,
AND
blot
Northern
band
time
In
in
BIOCHEMICAL
by
tures.
(data
the
1988
analyzed
longer
ccl
3,
of
the
establish
Sertol for
levels
can i
CRBP.
in
hormones
and
peri
The
both
that
cell
tubul
levels
to
ar
of
types.
known
the
the
Moreover,
affect
other
genes
process.
ACKNOWLEDGMENT This
work
was
supported
by
a
grant
from
the
MPI
(40%).
REFERENCES 1.
Fritz
I.B.
(1978)
Litwach 2.
In:
Biochemical
(Academic
Fritz
I.B.
testis,
Press,
(1985) vol
In:
123,
(Inserm, 3.
Wolbach
4.
Thompson
5.
Huang
6.
Gal
7.
Galdieri
8.
Galdieri
M.,
pp
S.B.
H.F. dieri
progress
Saez
hormones,
Vol.5,
Ed.:
A.,
Bertrand
G.
pp.249-279 in
J.M.,
cellular
Forest
endocrinology
M.G.,
of
Dazord
the J.
.15-56
and
Howe
J.N.,
of
York)
Recent
Eds:
Paris)
actions
New
P.R.
Howell
(1925)
J.,
Pitt W.C.
J
42,
Proc
(1979)
Biol
Reprod
Hembree
M.
and
Monaco
L.
(1983)
Cell
M.
and
Nistico
L.
(1986)
J
C.,
Med
(1964)
and
Caporale
Exp
G.A.
Adamo
Biol
S.
Rep
7,
(1986)
Sot
21,
Int
Androl
753-777 Roy
B
159,
510-535
891-904 7,
219-226
303-309
Molec
Cell
Endocr
48,
2 13-230 9.
Karl
A.F.
10.
Skinner
11.
Perez-lnfante
and
Mather 12.
J.P.
Griswold
V.,
Bardin
J.I.,
Griswold Hugly
S.and
14.
Chytil
F.
and
W.C.,
D.S.Goodman,eds),
15.
Kato
M.,
Sung
32,
173-189
16.
Porter
S.B., Androl
17.
Blaner
18.
Colantuoni
W.,
Galdieri U,
130,
D.E.,
Lee
Biol
and
Chytil
F.
Rich
McKnight
K.A.,
S.,
316-324
(M.B.Sporn, pp
Goodman and
D.C.,
121,
retinoids’ 9,vol.2,
K.
211-221 N.A.,
.89-l D.S.
23,
A.B.Roberts Academic
Biol
Press,
Reprod
Orgebin-Crist
M.C.
N.Y.
(1985) (1985)
197-212
V.,
Eriksson Comm
Ong 6,
Kato
27, Musto
332-340
Develop
chapter W.K.,
L.,
120,
(1984)‘The
1001-1003
383-392
Haywood
(1987)
D.E.
186, Reprod
G.L.,
118,
R.L.,
M.
Ong
Biol
Gunsalus
Endocrinology
Griswold
J
(1982)
Endocrinology
(1987)
and
Biochem
M.D.
ldzerda
M.
13.
M.D.(198D)
and
(1986)
Huggenvik
J
Griswold
M.K.
M., Cortese
Peterson
Goodman R., P .A.
D.
Nilsson and
(1987) M.,
Sundelin
431-439
1180
Biol
Lundvall J.
Reprod J.,
(1985)
36, Bavik
Biochem
130-137 C.O., Biophys
Res
Vol.
154,
19.
Dorrington 3,
20,
Hutson
21 .
Skinner
22.
Chirgwin
No.
Alvine 74)
24.
BIOCHEMICAL
J.H.,
Roller
N.F.
Stocco
D.M.
AND
and
BIOPHYSICAL
Fritz
I.B.
RESEARCH
(1975)
COMMUNICATIONS
Molec
Cell
Endocr
57-‘70 J.C.
Thomas Academic
and
M.F.
and
T.B.,
Bi ocheln 23.
3, 1988
is t ry
Griswold Przybyla
18,
J.C.,
(1981) M.D.
Endocrinology (1982)
A.E.,McDonald
Biol
108, Reprod
R.J.
1362-1369 27,
and
Rutter
211-221 W.J.
Kemp
D.J.
and
Stark
G.R.
(1977)
Proc
Nat
I Acad
53150-5354 P.
(1983) Press,
(1979)
5294-5299
In Orlando,
Methods
in
Enzimology
FL
1181
vol
100,
pp.255266,
Sci
USA