BIOLOGY
OF
43, 672-683
REPRODUCTION
(1990)
Characterization
of Sertoli
Cells
Relationship
between
and Polarized
MAKOTO
ONODA,2
Department
CARLOS
Cultured
Formation
Secretion
in the Bicameral
of Permeability
of Transferrin1
A. SUAREZ-QUIAN,
of Anatomy
and
Cell
DANIEL
Biology,
Washington,
DJAKIEW,
Georgetown
District
Chamber
Barriers
and
University
of Columbia
System:
MARTIN
Medical
DYM
Center
20007
ABSTRACT
cells
Sertoli
membrane
from
meability
barrier
subconfluent
tween
apical
fluid
levels
diffused
across
was
linear
relationship
higher
Is a function
(TI)
by
plated,
were
cells/well,
106
the
a few
apical/basal
were
Sertoli
believed
unique
cells
are known
by providing
apical
to be
(41-71
microenvironment
secreted
proteins,
of
than
a vital
were
the
located
epithelium,
of Sertoli
mic domains
not
only
in
are
Sertoli
cells
declined
plating
role
to
lack
1.57
and
layers
below
Received
March
‘Supported
density
and
(2)
[14-17].
acrosome
[2-7]
impermeable
When
2Correspondence:
in the
(M.D.), #HD23484
Department
Center, 3900
A
and
culture
Sertoli
this
cell
even
These
though
ratios
at 2.4
X
cells
those
indicate
epithelial
novo
of Tf
secretion
plated
results
de
amount
2 X 106 Sertoll
than
period,
most
total
monolayers
fewer
that
106
were
chambers
polarized
sheet.
crucial
nisms
secretion
for
of Anatomy
Reservoir
and
tro
[18,
is a result
of the
and of the ability
of
the
polarized
19]. Sertoli
chambers
with
cell
proteins
differentiation.
regulation
secretion
Hence,
of polarized
the role
of the
of the
obvious
by Sertoli
cells
is no doubt
the
mecha-
secretion
are
Sertoli
cells
difficulties
in vivo
and
in
in
in
a bicameral
chamber
system
was dethe study
of Sertoli
cell function
in vi-
cells grown
in this system’s
dual-commimic
more
closely
their
in vivo
respect
to both
structure
and
function
The bicameral
chamber
culture
system
has
proven extremely
useful for examining
fundamental
cell biological
phenomena
such as receptor-mediated
endocytosis [22], transcellular
transport
[20, 23], cell-to-cell
interactions [14, 15], and polarized
secretion
[4], events
that are
and #CA50229
Cell
of Sertoli
cell
conventional
culture,
veloped
to facilitate
of spe-
Biology,
Road, N.W., Washington,
germ
governing
studying
reside,
proteins
[1].
important
in establishing
spermatogenesis.
Because
of the
apical cytoplasit appears
that
(CAS.-Q.),
and spermatids
of Sertoli
cell
cells
to maintain
disparate
compositions
and basal compartments
of the seminiferous
Polarized
Recently,
SGPof developing
elaboration
germ
cells
secretion
[18, 20, 21].
Dr. M. Onoda,
University Medical
cm).
ohmS
resistance,
whereas
of an impermeable
epithelium
(D.D.)
Georgetown
(<33
in the
the
equilibrium).
of the Sertoli
the adluminal
andro-
compartment
as well as in the
cells [16]. Therefore,
#HD16260
chambers,
for
formation
meiotic
polarized
28, 1990.
Grant
layers
proteins,
but that some of these proteins
interact
with
developing
germ cells. Significantly,
the unique
microenvironment
of the adluminal
compartment,
within
which
8, 1990.
by NIH
when
the apical
and
chamber
when
0.6%
of added
[3Hlinulin
Conversely,
the electrical
differences
of cells.
phenotype
June
be-
is defined
electhcal
and
However,
I in a 24-h
partment
Accepted
flux
significant
observed.
of hydrodynamic
in spermato-
(TI)
adluminal
involved
inulin
No
were
2.03
gen-binding
protein
(ABP)
[3, 4, 7, 8], plasminogen
activator [9, 10], and inhibin
[11]. In addition,
Sertoli
cells secrete a 70-kDa monomeric
protein
(SGP-1)
and a dimeric
protein
(SGP-2)
of 81 kDa [12, 13], two immunologically
distinct
proteins
that may regulate some aspects
of germ
spermatids
seminiferous
transport
monolayer
permeable
in the
of bicameral
medium
cells
microenvironment
transferrin
development
in the
than
between
chamber.
1.0 in permeable
by the
(1)
basal
of the
of Sertoli
Tf secretion
on
in the
period
less
(recognized
luminal
cell differentiation
and
1 was
immunolocalized
exists
3)
and
cific
to play
including
cm2)
ohmS
0.808)
[3Hjinulin
of fluid levels
between
the two sides
of the
7.5 ±
cultures.
a per-
formed
(permeable),
and
confluent
basement
that
barrier
resistance,
impermeable
reconstituted
cultures
a permeability
Sertoli
cell monolayers,
± 0.5% In permeable
13.7
for germ cell differentiation
[1]. l’his
is formed
in part by Sertoli
cell-
essential
form
with
monolayer
electrical
An
confluent
reservoir
collection
but
is dependent
a unique
impregnated
hydrodynamic
equilibration
barrier
is present
between
INTRODUCTION
genesis
not
equilibrium,
versus
detected
the
monolayers
a 15-20-h
polarity
cells
filters
confluent
1)
did
examined.
r =
+ 91.50,
proteins
permeable
monolayers
by Sertoli
that
fluid
able to prevent
is, a permeability
monolayers
into
respectively,
impermeable
secretion
among
Millipore
obtaIned:
density.
of
or
on
were
cultures
chambers
-4.68(X)
secreted
of Tf during
contained
of the
=
of cell
impermeable
(apical/basal)
X
relationships
Impermeable
(Y
one
was
proteins
3.6
the
in
relationship
Transferrin
secreted
reservoirs
cultured
monolayer
confluent
The
were
of cultures
(volumes)
do not change.
In the impermeable
the monolayer
during
a 6-h collection
period
reciprocal
and
basal
old)
types
Sertoli
cell epithelial
sheet
are
of a bicameral
chamber.
That
resistance
synthesized
2)
(permeable).
and
days
Three
(impermeable),
the cells of the
basal reservoirs
(18
chambers.
cultures
the
rats
Immature
in bicameral
DC
20007.
672
SERTOLI
to occur
in the seminiferous
oratory
recently
demonstrated
that
are secreted
in a polarized
manner
known
cameral
inhibin
culture
secretions
than in the
ber
chambers
are four
basal
chamber,
cal/basal)
Janecki
of Tf secretion
and Steinberger
mostly
in
ancies
have
the biology
ther
the
basal
[15].
times
CULTURE
IN
epithelium.
Our labSGP-1 and SGP-2
by Sertoli
cells in bipolarities
of ABP and
both
The
higher
in the
apical
the
polarity
whereas
is only 1.5-2
[24] reported
compartment
CELL
cham-
(api-
[4, 11]. In contrast,
that Tf is secreted
Clearly,
important
implications
in our
of the seminiferous
epithelium
these
discrep-
understanding
and require
of
fur-
clarification.
In this
titatively
grown
report,
we
address
the impermeability
in the bicameral
permeability
hydrodynamic
the
need
to characterize
quan-
of Sertoli
cell epithelial
chambers.
We correlate
of confluent
Sertoli
equilibrium,
cell
electrical
flux. Our definition
of impermeability
vention
of hydrodynamic
equilibration
epithelial
resistance,
depends
of culture
sheets
the im-
sheets
with
and
inulin
on the
media
prebe-
tween
the apical
and
basal
reservoirs
of the bicameral
chambers.
We report
that once Sertoli
cells reach
100%
confluency,
the formation
of an effective
permeability
barrier is dependent
upon cell density.
Tf secretion
by Sertoli
cells is intrinsically
bidirectional
in vitro. However,
only at
higher
cell densities
do we observe
polarized
secretion
of
If in the apical
direction,
a condition
that may resemble
the physiological
state of Sertoli
cells in the seminiferous
epithelium
facilitating
the generation
of the special
milieu
in the adluminal
compartment.
MATERIALS
Isolation
and
Culture
Sertoli cells were
ley rats as described
AND
of Set-toll
METHODS
Cells
isolated
from 18-day-old
Sprague-Dawpreviously
by Hadley
et al. [25], with
only slight modifications.
Briefly, the decapsulated
testes were
minced
into small fragments
and incubated
in Dulbecco’s
Modified
Eagle’s Medium
(DMEM;
Irvine
Scientific,
Santa
Ana, CA) containing
trypsin
(1.5 mg/mI)
and DNase
(20 g/
ml), for 15 mm at 37#{176}C
in a shaking
water bath (90 oscillations/min)
to remove
Leydig cells and other interstitial
tissue.
The tubule
fragments
were washed
with 10% fetal
bovine
serum
(FBS)/DMEM,
followed
by a wash with DMEM
alone. Subsequently,
the seminiferous
tubules
were treated
with a second
enzyme
solution
containing
collagenase
(2
mg/mi;
Worthington
Biochemical
cOrp.,
Freehold,
NJ),
hyaluronidase
(2 mg/mI),
and DNase
(20 p.g/ml)
in DMEM,
for 30 mm at 37#{176}C
in a shaking
water
bath to remove
the
peritubular
cells. During
the second
enzyme
digestion,
fragments
were agitated
every
10 mm by aspiration
with a
Pasteur pipette.
To ascertain
effectiveness
of the second
enzyme digestion,
removal
of peritubular
cells was monitored
by examining
aliquots
of the digestion
with an inverted
phase-contrast
microscope.
At this point,
aggregates
contained
approximately
50 or more
Sertoli
cells. The cell
BICAMERAL
673
CHAMBERS
per aggregate
was determined
by treating
cells with
a trypsin-versene
solution
(Bioproducts,
Inc., Walkersville,
MD) and counting
the cells using a Hausser
HY-Lite corpuscle-counting
chamber
with an Olympus
BH-2 microscope. This aggregate
suspension
was plated
on Matrigel”
(Collaborative
Research
Inc., Lexington,
MA)-coated
filters
in bicameral
chambers
(Millicell”-HA;
Millipore
Corp.,
Bedford,
MA) at 2.4 X 106 or 3.6 X 106 cells/0.64
cm2/well
as described
in Byers et al. [18] and cultured
with serumfree defined
medium
(SFDM)
[25] for 5 days at 34#{176}C
in a
mixture
of 5% C02/95%
air until monolayers
formed.
Sertoli cells plated
after this step would
at times give rise to
“leaky”
(permeable)
chambers
in which
hydrodynamic
equilibration
of culture
media was never eliminated
during
the culture
period.
Alternatively,
the tubules
were digested
with a third enzyme solution,
containing
trypsin
(0.5 mg/mI),
EDTA (0.1
mg/ml),
and glucose
(4 mM) in Ca2-,
Mg2-free
PBS, for
number
15 mm
at 37#{176}C
in a shaking
water
bath
to obtain
smaller
or less). The preparations
were
washed
with 10% FBS/DMEM
then with DMEM alone. The
Sertoli
cells were counted
as described
above and plated
at concentrations
varying
from 1.0-3.6
X 10 cells/0.64
2/
well. Medium
was replaced
every two days with fresh SFDM,
and the cultures
were continued
for 5 days until used in
the experiments.
SFDM consisted
of DMEM supplemented
with 100 U/mI
penicillin,
0.25 pg/ml
fungizone,
100 p.g/ml
streptomycin,
100 ng/ml
FSH, 2 pg/ml
insulin,
10 ng/ml
epidermal
growth
factor, 5 .tg/ml
human
Tf, 50 ng/ml
vitamin
A, 200 ng/ml
vitamin
E, i0
M hydrocortisone,
i0
M testosterone,
108
M estradiol,
2 mM glutamine,
5 ng/ml
sodium
selenate,
1
mM sodium
pyruvate,
22 mM sodium
lactate,
and 3 pg/ml
cytosine
arabinoside.
tubules
10 cells
(containing
Determination
of Electrical
Resistance
with SFDM for 5 days, the transepithelial
resistance
of Sertoli
cells was measured
by an
impedance
meter (EVOM; World Precision
Instruments,
New
Haven,
CT) using alternating
current
to make electric
resistance
measurements
as described
by Cuthbert
et al. [26].
The Sertoli cell cultures
were then examined
for their ability to impede
passage
of inulin-[3H]methoxy
(250 tCi/054
mg; ICN Biomedicals
Inc., Costa Mesa, CA) from the basal
to the apical reservoir
of the bicameral
chambers
as described
below.
The net value of electrical
resistance
was
computed
by subtracting
the background,
which was measured on Matrigel-coated,
control
cell-free
chambers,
from
values
of Sertoli
cell-plated
chambers.
Finally,
the values
were multiplied
by 0.64, the effective
area of the filter in
the apical chamber.
After
culture
electrical
Permeability
Studies
The permeability
characteristics
of Sertoli cell monolayers
were
of the epithelial
sheets
determined
by measuring
674
ONODA
the free diffusion
of [3H]inulin
[18]. The Sertoli cell monolayers were used for transport
studies
after electrical
resistance was determined
as described
above.
Culture
chambers containing
400 p1 of SFDM in the apical
compartment
were placed in a 24-well multiwell
plate containing
400 p1
of SFDM with I pCi of [3H]inulin.
After 2, 4, and 6 h of
incubation
at 34#{176}C,
10-pl aliquots
of medium
were
collected from each compartment,
and the distribution
of radioactivity
was determined.
The radioactivity
of transported
[3H]inulin
in the apical compartment
was represented
as a
percentage
of total recovered
radioactivity.
The percent
yield
of radioactivity
was 93.1 ± 1.1% in six different
experiments
(56 total chambers).
The inulin
transport
in the
chambers
with Matrigel-coated
filter alone was 14.0 ± 1.1%
(n = 18), 25.4 ± 1.3% (n = 18), and 31.4 ± 0.9% (n =
21) during
2, 4, and 6 h of incubation,
respectively.
Determination
of Tf
The amount
of If secreted
by Sertoli cells into the culture media was determined
by ELISA using Bio-Rad (Richmond,
CA) alkaline
phosphatase
kits [15,27].
The antibody
(Cooper
Biomedical,
Malvem
PA) used in the ELISA
was
directed
against
rat serum
Tf. The sensitivity
of the assay
was at least 3 ng per assay well, and the detectable
crossreactivity
in fresh SFDM was less than 3 ng per assay well.
Conditioned
media
were collected
from both apical and
basal
reservoirs
at various
times (as indicated
in the legends of the tables and figures)
after determination
of electrical resistance
and inulin flux on Day 5. Subsequently,
the
concentrations
of If in each well were measured
separately
to determine
the apical/basal
ratio as a measure
of polarized If secretion,
The amount
of Tf secreted
into each compartment
was obtained
by multiplying
the concentration
of
Tf by the volume
of each of the compartments.
The apical!
basal ratio was computed
from the sum of Tf in each compartment.
The total amount
of secreted
If from Sertoli cells
was obtained
by the addition
of both amounts.
The amount
of secreted
If was indicated
as pg/well,
because
the cell
numbers
before
and
in the experimental
Protein
Labeling
after
culture
with
culture
period
varied
by less
than
10%
[4].
[35S]Methionine
Proteins
synthesized
de novo by Sertoli
cells were labeled with [35S]methionine
(ICN) according
to the method
of Djakiew
and Dym [15]. Briefly,
impermeable
confluent
Sertoli
cell monolayers
were washed
with DMEM twice and
precultured
with 90% methionine-deficient
SFDM (1/10 MetSFDM)
for 6 h. Then both the apical and basal media were
replaced
with
fresh
1/10
Met-SFDM,
and
50
[35S]methionine
was added
to the basal medium.
tioned
media
were
collected
from the apical
chambers
separately
after a 24-h culture period
centrated/dialyzed
with Centricon
P-b (Amicon
Grace
& Co., Danvers,
MA). After radioactivities
pCi
of
Condibasal
and conDiv., W.R
and
were
de-
El AL.
termined,
labeled
proteins
were separated
by SDS-PAGE
and the protein
bands
were observed
by fluorography
Immunoprecipitation
of [35S]Methionine-Labeled
[28],
[29].
Tf
[35S]Methionine-labeled
Tf was immunoprecipitated
by
the Protein A-Sepharose
(Pharmacia
Inc., Piscataway,
NJ) antibody complex
method
according
to Kessler
[30]. Five microliters
of rabbit anti-rat iT (Cooper
Biomedical)
was added
to a 25-pl Protein
A-Sepharose
suspension
and incubated
for 3 h at 4#{176}C.
The Protein
A-Tf antibody
complex
was further incubated
overnight
at 4#{176}C
with radiolabeled
sample
protein
(100
pA) in which
nonspecffically
bound
proteins
had been previously
removed
as described
in the method
of Sylvester
et al. [31]. The immunocomplex
was washed
five times with PBS containing
0.5% Tween
20/0.1%
BSA
and solubilized
by the appropriate
electrophoresis
sample
buffer at 100#{176}Cfor 4 mm. Subsequently,
the immunoprecipitated
PAGE,
[35S]methionine-labeled
the
by fluorography.
and
specific
Tf was
immunoprecipitate
separated
was
by SDSvisualized
Chemicals
and
17) was a gift from
the
of the National
Institutes
of Health (Bethesda,
MD).
All other
chemicals
were purchased
from Sigma Chemical
Co. (St. Louis, MO) unless
indicated
otherwise
in the text.
FSH
(NIADDK-0FSH-16
NIADDK
Statistics
Statistical
analyses
for comparison
ered significant
were
performed
with
of two groups.
Differences
when p < 0.05.
Student’s
were
t-test
consid-
RESULTS
Hydrodynamic
Equilibrium
of Sertoli Cell Monolayer
as a Measure
Permeability
At the
beginning
of Sertoli
cell culture,
equal volumes
of media
were added
to each compartment
of the bicameral chamber.
During
the first two days after initial plating
of the cells, the media
in the apical
and basal reservoirs
attained
hydrodynamic
equilibrium.
That is, the meniscus
of the medium
in the apical
reservoir
reached
the same
level as the meniscus
of the medium
in the basal reservoir.
This equilibrium
was attained
within
12 h even if unequal
volumes
were added
to each reservoir
of the bicameral
chamber.
Once
the
rier to free diffusion
our culture
conditions),
ibration
was abrogated.
then
added
ervoir,
the
out
the
tected
medium
to the
unequal
length
in
of the
Sertoli
cells
of protein,
formed
(usually
a permeability
after 2-3
days
however,
the hydrodynamic
For example,
if more
medium
apical
volume
culture
reservoir
distribution
(Fig.
than
IA).
to the
remained
This
was
the bicameral
chambers
by adding
to reach the rim of the apical
reservoir.
barin
equilwas
basal
resthroughreadily
de-
sufficient
In these
SERTOLI
A:
Impermeable
B
Permeable
CELL
CULTURE
IN BICAMERAL
CHAMBERS
675
Layer
Layer
FIG. 1. Schematic
diagram
of the bicameral
culture chamber
system
used to grow sheets of Sertoli cells in which an effective
permeability
barrier is
either present
(A), or not present
(B). Culture
medium
of the apical reservoir
(400pi)
and the basal reservoir
(400 p.1) attained
hydrodynamic
equilibration
after 15-20
h of culture
in the permeable
layers (B), i.e. 296 ± 7 p.1 and 472 ± 5 p.1 In = 17) of cultured
medium
were recovered
in the apical reservoir
and in the basal reservoir,
respectively.
In A, when the fluid level was increased
to the rim of the inner chamber,
a convex
meniscus
formed
that was
detected
readily
with
the
naked
eye.
ONODA
676
cases, the medium
of the apical reservoir
formed
a convex
meniscus
that was observed
with the naked eye. In contrast,
in permeable
layers of Sertoli cell sheets, the height of the
medium
within
the apical reservoir
equilibrated
with the
height of the medium
in the basal reservoir,
attaining
hydrodynamic
equilibrium
(Fig. 1B). For instance,
in chambers exhibiting
hydrodynamic
equilibrium,
the initial 400
p1 in the apical reservoir
decreased
to 296 ± 7 p1, and the
basal reservoir
medium
increased
proportionally
in volume to 472 ± 5 pA (n = 17) during
a 15-20-h
period.
In
contrast,
no change
in fluid volumes
was detected
in bicameral
chambers
not maintaining
hydrodynamic
equilibrium that had established
an effective
permeability
barrier.
These qualitative
observations
with the naked eye make a
reliable
and rapid assay for detecting
the formation
of impermeable
epithelial
sheets
of Sertoli
cells in bicameral
chambers,
at least regarding
fluid-hydrodynamic
equilibrium.
The frequent
absence
of a permeability
barrier
of Sertoli
cell epithelial
sheets was correlated
with cultures
of Sertoli
cells plated from larger aggregates
containing
appoximately
50 or more Sertoli cells obtained
after the second
enzyme
digestion
(see Materials
and Methods).
A likely explanation
of this phenomenon
is that larger cell aggregates
do not
spread
evenly
but produce
an uneven
layer, with multilayers in some part of the filter surface.
Therefore,
a third
enzymatic
digestion
was performed
on the large Sertoli cell
aggregates
to generate
clumps
containing
approximately
10
Sertoli cells. Use of these smaller
aggregates
as seeding
material generated
Sertoli cell cultures
that rarely failed to establish a permeability
barrier
as determined
by the hydrodynamic
properties
of the media
in the reservoirs
of the
bicameral
chambers.
Subsequent
culture
experiments
to
examine
the polarity
of Sertolm cell secretion
used these
smaller
aggregates,
which
form
impermeable
epithelial
sheets.
Relationship
between
Electrical
and [3HJtnulin
Transport
ET
AL.
examined.
These data are presented
in Table 1. The electrical resistance
was 52.2 ± 1.4 ohm
cm2 and 62.9 ± 5.0
ohm
cm2 in impermeable
epithelial
sheets plated at 2.4 x
106 cells/well
and 3.6 X 106 cells/well,
respectively.
These
values are 2-3 times higher than those of permeable
layers
lacking an effective permeability
barrier. The flux of [3H]inulin
transport
in a basal-to-apical
direction
in impermeable
epithelial
sheets
during
a 6-h incubation
period
was 8.4 ±
0.3% and 6.6 ± 0.6% at 2.4 x 106 cells/well
and at 3.6 x
106 cells/well,
respectively.
These values are about two times
lower than the values obtained
in the permeable
layers. Every
individual
value represented
in Table 1 is shown
in Figure
2. The regression
line of the electrical
resistance
on inulin
transport
is Y = -4.68(X)
+ 91.50
(correlation
coefficient
r = 0.808). Culture
wells were judged
to contain
impermeable epithelial
sheets if a lack of hydrodynamic
equilibrium
was maintained.
Thus, in Figure
2, one set of values
for
[3H]inulin flux was judged to represent
an impermeable
and
a permeable
layer of Sertoli cells because
of the absence
and presence,
respectively,
of hydrodynamic
equilibrium.
Effect of Cell Density
on Inulin
and Electrical
Resistance
Transport
As the
plating
density
of Sertoli
cells in the chambers
the flux of [3H]inulin
transport
increased
(Fig. 3A).
Inulin
transport
was 9.8 ± 0.7%, 7.3 ± 0.4%, and 5. ±
0.1% (average
7.5 ± 0.6%) during
a 6-h incubation
period
at 1.2 X 106 cells, 2.4 x 106 cells, and 3.6 X 106 cells/well,
respectively.
The flux of [3H]inulin
in permeable
epithelial
sheets (13.8%)
represented
by the broken
line in Figure 3A
was much greater
than in impermeable
epithelial
sheets of
Sertoli cells at the lowest cell density (1.2 x 106/well).
The
regression
line of the electrical
resistance
versus inulin flux
was Y = -5.87(X)
+ 100.23
(r = 0.911) (Fig. 3B). This
difference
in the regression
lines between
Figure
2 (Y =
-4.68(X)
+ 91.50) and Figure
3B reflects
the inclusion
of
values of permeable
epithelial
sheets in the data presented
decreased,
Resistance
Initial morphological
studies of Sertoli cells in vitro, prepared as described
in Materials
and Methods,
seeded
at 2.4
X
10 cells/well,
and grown
in bicameral
chambers,
revealed that the entire growing
surface
of the culture
wells
was covered
by the Sertoli
cell layer. However,
free diffusion of macromolecules,
revealed
by measuring
the flux of
[3H]inulin
across
the Sertoli
cell epithelial
sheet, was frequently
observed
in these cultures.
Moreover,
the absence
of a permeability
barrier
occurred
frequently
even when
greater
concentrations
of cells (3.6 X 10 cells/well)
were
used during
initial seeding
of the cultures.
To characterize
quantitatively
the confluent
epithelial
sheet
of Sertoli
cells grown
in bicameral
chambers
with respect
to the generation
of a permeability
barrier,
the relationship
between
electrical
resistance
and [3H]inulin
transport
was
TABLE
1.
Relationship
between
Cell density
(x105 cells/well)
Barrier
formationb
2.4
2.4
3.6
3.6
+
-
+
-
In
In
=
In
(n
=
=
electrical
resistance
(3HJlnulin
transport’(%)
19)
9)
16)
12)
8.4
14.4
±
±
6.6 ±
13.2
±
0.3*
0.6
0.6*
0.6
and inulin
transport.
Electrical
resistanced
(ohm
cm2)
52.2
16.5
62.9
32.9
±
1.4*
±
4.5
5.0*
1.3
±
±
Sertoli cells obtained
after second or third enzyme
digestion
were plated
in the apical chamber
(0.64 cm2) of bicameral
chambers,
and cultured
for
5 days; then, electrical
resistance
and inulin flux were measured.
Media
were replaced
by fresh SFDM (400 p.1 in each compartment)
and cultures
were continued
for 15-20
h for measurement
of Tf secretion.
blmpermeable
epithelial
sheets (+); permeable
epithelial
sheets (-).
cValues represent
mean ± SE of percent of total radioactivity
obtained
from
3 different
experiments.
dvalues
represent
mean
±
p < 0.01 (statistics
between
in each set of cell density).
SE.
impermeable
1+] and
permeable
1-)
sheets
SERTOLI
CELL
CULTURE
IN BICAMERAL
CHAMBERS
Transferrin
Secretion
677
by Sertoli
Cells
In those
Sertoli cell cultures
where
electrical
resistance
permeability
were measured
(described
above),
the amount
of If secreted
was also determined
(Table
2).
The total Tf secretion
by Sertoli cells was not significantly
different
between
the impermeable
epithelial
sheets
and
the permeable
epithelial
sheets,
although
the Tf secretion
was slightly
less in the permeable
layers. However,
the polaity of the If secretion
by the epithelial
sheets
during
a
15-20-h
collection
period
was significantly
higher
for the
impermeable
epithelial
sheets
than for the permeable
epithelial
sheets.
The apical/basal
ratios were 2.03 and 1.57
and
I
.
Ys-4.68#{149}X
+ 91.50
(r0.808)
i,.
E
S
0
E
for the impermeable
cell layers, and 0.78 and 1.12 for
permeable
layers plated
at cell densities
of 2.4 x 106
3.6 X 106/chamber,
respectively
(Table
2). Although
apical/basal
ratio was greater
for 2.4 x 106 cells/well
.
0
.
0
0
C
0
0
0
0
0
U
S
I-
0
.#{149}\
e
a
w
o0
0
Or,..0
0
‘
0
0
0
00\\
0
0
5
lnulin
10
Transport
inulin
15
( S of
total
20
count
)
FIG. 2. Relationship
between
the electrical
resistance
and inulin transport in the cultured
Sertoli cells. The dots represent
the values obtained
individually
in the same experiment
described
for Table 1. The regression
line between
electrical
resistance
and inulin transport
is V = -5.51(X)
+
97.39
(r = 0.912) and V = -4.64(X)
+ 94.21(r
=
0.906)
at 2.4 x 106 cells/
well In = 28) and 3.6 x 106 cells/well
In = 28), respectively.
(See Materials
and Methods
for further details regarding
culture conditions.)
#{149}:Impermeable layers. 0: Permeable
layers.
in Figure
2, whereas
all cultures
presented
in Figure
3B
maintained
impermeable
epithelial
sheets.
Cell plating
density also exerted
a marked
effect on Sertoli cell morphology
(Fig. 4). Ax low cell density,
layers of
Sertoli cells appeared
more cubical.
Increasing
plating density of cells to 2.0 x 106 cells/well
led to an enhanced
development
in the columnar
morphology
of the Sertoli
cells
in vitro. Increasing
the plating density beyond
2 x 106 cells/
well led to a marked
increase
in the number
of nuclei per
unit area and in transformation
of nuclear
morphology
of
Sertoli
cells. The nuclei became
more elongated
and gained
a orientation
perpendicular
to the base of the growing
Sertoli cells. The significance
of these shape changes,
however,
is not known
at this time.
the
and
the
than
for 3.6 x 106 cells/well
(Table 2), there was no significant
difference
between
densities
according
to Student’s
t-test
(p > 0.05). These results suggest
that estimates
of electrical
resistance,
inulin transport,
the polarity
of Tf secretion,
and
the retention
of medium
in the apical chamber
with time
depend
on the formation
of an effective
permeability
barrier by epithelial
sheets.
Next, we examined
the cell-plating-density-dependent
Tf
secretion
by Sertoli cells in greater
detail (Table 3). During
an initial 24-h collection
period,
as expected,
Tf secretion
by Sertoli cells increased
as the cell plating
density was increased.
However,
this density-dependent
secretion
of If
by Sertoli
cells was not observed
during
a 48-h collection
period.
The secretion
ratio (apical/basal)
of Tf increased
during
a 24-h incubation
period
as the number
of plated cells increased
(Table
3 and Fig. 5). In the chambers
plated
with
Sertoli
cells, at lower cell density,
however,
polarized
secretion
of Tf was not observed
even though
impermeability
of the epithelial
sheets,
as judged
by hydrodynamic
equilibrium,
was present
in the chambers.
Detection
of polarized If secretion
by Sertoli cells required
that plating
density per well reach at least 2 x 106 cells/well.
However,
in
each culture
well that failed to exhibit
polarized
Tf secretion, morphological
examination
revealed
cells lacking
TABLE
2. Tf secretion
chamber
system.
Cel I density
(x 106 cells/well)
2.4
2.4
3.6
3.6
by Sertoli
cells
grown
Transfer
Barrier
Total
formation
+
-
+
-
In
(n
(n
In
in the bicameral
(p.g/well)
19)
0.440
±
0.0486
6)
0.383
±
=
16)
0.582
±
=
11)
0.443
±
0.013
0.033
0.072
=
=
culture
nfl secretion
Ratio
2.03
0.78
1.57
1.12
(apical/basal)
±
0.20*
0.03
0.17*
±
0.09
±
±
impermeable
epithelial
sheets 1+); Permeable
epithelial
sheets I-)
‘The
values represent
mean ± S.E. of total Tf secreted
into both apical
and basal medium
for 15-20 hr.
CThese values represent
mean ± S.E.
p
< 0.01, * p < 0.05 (statistics
between
impermeable
(+) and permeable
I-I sheets
in each
set of cell density).
678
ONODA
ET
AL.
100
-
80
B
-
0
Y=-5.87
X
+
100.23
c,i
E
C
C rQ.911
)
C)
0
(I)
0
E
0
4-
0
0
60
0
-
©
©
0
a’
©
C)
C
Ca
6..
CO
0
0.
(0
0
a,
C
40
(6
-
I-
I-
Ca
C.)
C
I-
0
C
a,
w
20
2
Incubation
FIG. 3. (A)
(1.2 x ion, 2.4
sample
harvest
± SE obtained
-
4
Time
10
7.5
5
( hr )
Inulin
Transport
C % of total
count
)
Inulin transport
on Sertoli cells grown in bicameral
chamber
system.
Sertoli cells were plated in the apical chamber
at different
densities
x 10, and 3.6 x 10 cells/0.64
cm’/weIl),
and cultures
were carried
out according
to the schedule
described
for Table 1 except
for the
time. The results presented
here are from cultures
in which impermeable
monolayers
had been established.
The values represent
mean
from 4 different
wells. The broken
line represents
the values detected
in the permeable
layers described
in Table 1.
(B) Relationship
between
inulin transport
and electrical
exactly as described
for Table 1 and Figure 34. The values
x 106 cells/well;
#{174}:1.2x 106 cells/well.
resistance
in Sertoli
of inulin transport
characteristic
features
of polarized
Sertoli cells in vivo; i.e.,
cells resembled
those in the top two panels of Figure 4. In
addition,
polarized
Tf secretion
by viable Sertoli
cells, assessed by morphological
means, gradually
declined
during
a 48-h incubation,
even in cultures
plated
at higher
cell
densities
(Table 3 and Fig. 5).
The secretion
of Tf by Sertoli
cells was also examined
by labeling
de novo-synthesized
proteins
with [35Sjmethionine
and determining
the secreted
protein
profiles
by fluorography.
De novo synthesis
and secretion
of Tf were compared
to those of other Sertoli
cell proteins.
The majority
cells
were
grown
obtained
of de novo
in the
from
bicameral
chamber
system.
incubation
period. 0:3.6
a 6-h
Culture
conditions
x 106 cells/well;
were
#{174}
:2.4
proteins
were secreted
into the api6), although
three
major bands
(>200
kDa, 67 kDa, and <14 kDa) were also secreted
in significant quantities,
along with other faint bands (34-4 1 kDa),
into the basal
chamber.
Immunoprecipitation
of labeled
proteins
with a specific antibody
to rat If indicated
that the
67-kDa band corresponded
to If. This is consistent
with the
initial
report
on the molecular
mass of rat Tf [32]. Importantly, it should
be noted
that the overall
secretion
of If
does not parallel
the remarkable
polarized
secretion
observed
for the majority
of other
Sertoli
cell proteins.
Alcal
chamber
synthesized
(Fig.
1.0
io6 cells/well
x
I
1.5 x io6 cells/well
2.0 x io6 cells/well
2.4 x io6 cells/well
3.6
x io6
cells/well
--.,.
FIG. 4. Morphology
described
in Materials
Nine days after plating,
of Sertoli cells in vitro plated at different
densities.
Sertoli
and Methods
and plated at different
cell concentrations
per
cells were prepared
for routine
light microscopic
observation.
cell cultures
were prepared
as
well as indicated
in the figure.
x800.
680
ONODA
I
1.5
I
I
El
AL.
I
ABCD
24hr
-
-
C
0
92 k
48 hr
4-
0
6.
C,
a
0)
1.0
mTf
-
IS
0
4
(6
a
(6
.0
46
k1
(6
U
0.
(6
0
4-
0.5
-
I
ii
6
Ratio
C x106
DensIty
of transfernn
(apical/basal)
at different
cell densities.
described
in Table 3.
-
2
1
l
Cell
FIG. 5.
i.
30 k
(6
These
values
TABLE
3
4
/well
secretion
were
3.
FIG. 6. Fluorography
of immunoprecipitation
for If from both apical
and basal
media in the bicameral
chamber
system.
Sertoli cells (2 x 106
cells/well)
were cultured
with SFDM containing
I’5S]methionine
for 24 h,
and conditioned
media were harvested
at the termination
of culture.
Lanes
A and B, obtained
after 9 days exposure,
represent
crude
conditioned
medium from
the apical and basal reservoirs,
respectively.
Lanes C and D,
obtained
after 1 day of exposure,
represent
Tf immunoprecipitated
from
the apical and basal medium,
respectively.
derived
Tf secretion
by Sertoli
from
cells plated
the experiment
by impermeable
0-24
Cell density
(x
10
cells/well)
1.0
1.5
2.0
2.4
3.6
4Cuftures
±
were
from
SE obtained
collected
(p.g/well)
If
continued
though
significant
amounts
of Tf are secreted
into both reservoirs
by Sertoli
cells in vitro, it should
be noted
again
that a greater
polarized
secretion
of If was observed
in Sertoli cell cultures
of higher
density.
At this time,
however,
we do not know the identity
of the two other major proteins secreted
basally.
)
Sertoli
cell sheets
at different
h
cell densities.4
0-48
Ratio
(A/B)
0.172
±
0.022
0.85
±
0.304
0.383
±
0.85
±
±
1.00
±
0.380
±
1.10
±
0.448
±
0.020
0.022
0.051
0.055
1.44
±
for 48 h without
plated
medium
both the apical and basal chambers
from four different
wells.
0.25
0.12
0.14
0.20
0.20
exchange.
Tf (p.9/well)
0.417
±
0.040
0.495
±
0.043
0.616
±
0.067
0.480
±
0.088
0.607
±
0.085
After
for Tf measurement.
24 h, 3O-p.l
Values
h
Ratio
0.71
0.86
0.80
0.97
1.20
aliquots
represent
(A/B)
±
0.05
±
0.08
±
0.06
±
0.10
0.21
±
were
mean
SERTOLI
CELL
CULTURE
DISCUSSION
A principal
feature
of many epithelial
cells is their intrinsic ability to form focal adhesion
sites at points of contact with one another
and thus create
a permeability
barrier. By creating
such a permeability
barrier,
epithelial
cells
establish
distinct
domains
at their apical and basolateral
cell
surfaces
that lead to differential
expression
of structural
and
functional
components.
This fundamental
property
of Sertoli cells within the seminiferous
epithelium
is manifested
in the elaboration
of polarized
structural
and functional
features such as protein
secretion
[4,6,7,9,
11, 16]. In this report, we characterize
further
the parameters
that govern
the polarized
expression
of Sertoli
cell function
in dualcompartment
culture.
In a previous
in vitro study from our laboratory
[20] using Sertoli cells from 10-day-old
rats, we demonstrated
the
correlation
between
electrical
resistance
and formation
of
a permeability
barrier
in a confluent
monolayer
of Sertoli
cell epithelial
sheets after 1-2 days in culture.
The formation of classical
Sertoli
cell tight junctional
complexes
as
revealed
by morphological
means, however,
required
at least
5 days of culture
[20]. In this study, the electrical
resistance
increased
on Day 1-Day
2 and temporarily
decreased
to
basal levels during
cell-remodeling.
The electrical
resistance increased
again and reached
a plateau
at Day 5. This
second
increase
in electrical
resistance
was maintained
for
at least the next 3-4 days. The initial increase
of electrical
resistance
probably
was due to cell-cell communication
and
occurred
before
cell-remodeling
brought
about by the formation
of rigid barriers
(classical
tight junctions).
In the
present
study, we report
data from cell cultures
obtained
from 18-day-old
rats. Electrical
resistance
of the Sertoli cell
epithelial
sheets
correlates
inversely
with inulin transport
and serves
as a reliable
indicator
of the formation
of an
effective
permeability
barrier.
That is, inulin
flux occurs
readily,
whereas
electrical
resistance
decreases,
in permeable layers of Sertoli
cells; in impermeable
layers the reverse phenomenon
takes place.
Although
radioactive
substances
such as [3H]inulin
[6, 18]
or [atRb]Cl [33] are commonly
used to estimate
permeability barriers
formed
by Sertoli
cell monolayers,
contamination of Sertoli cell epithelial
sheets with radioisotopes
is a
major disadvantage
of this method.
In contrast,
the relative
ease of measuring
electrical
resistance
and the lack of cell
layer contamination
with radioisotopes
in our method
makes
it an ideal means
of monitoring
cellular
confluency
and impermeability
in bicameral
chambers.
Moreover,
we report
a simple
but reliable
test for impermeabffity
of confluent
epithelial
sheets that requires
only
observation
of the bicameral
chambers
with the naked eye.
Once the Sertoli cell epithelial
sheets establish
a diffusion
barrier,
hydrodynamic
equilibration
of media between
the
two chambers
ceases to exist. This phenomenon
can be detected by adding
sufficient
medium
to the apical reservoir
IN BICAMERAL
CHAMBERS
681
so as to create a convex
meniscus.
In impermeable
epithehal sheets,
this convex
meniscus
is maintained
during
the
duration
of the cultures
and correlates
with low inulin flux
and enhanced
electrical
resistance.
Generation
of a Sertoli
cell model
system
in vitro that
maintains
the in vivo phenotypic
expression
of the cells facilitates
the further
examination
of polarized
protein
secretion
by Sertoli
cells. Characterization
of this system is of
fundamental
importance
because
equivocal
results
have been
presented
in the literature
regarding
the polarized
secretion of Tf by Sertoli
cells. For example,
in earlier
reports
by Janecki
and Steinberger
[19, 24], the apical/basal
ratio
of If secretion
in the two-compartment
culture
chamber
system was reported
to be 0.20-0.56.
In addition,
the ratio
of ABP secretion
was 1.43 [24], whereas
our laboratory
reported
a ratio of 4.0 [4]. Significant
differences
between
the
experimental
conditions
of Janecki
and Steinberger
[19, 24]
and of our present
study may account
in part for this discrepancy.
In particular,
the apical/basal
ratio of less than
1.0 for Tf secretion
reported
by Janecki
and Steinberger
[24] may be related
to the hydrodynamic
equilibration
of
fluid volumes
across their bicameral
chamber.
As demonstrated in the results presented
in lable
3 and Figure 5, the
polarity
of If secretion
by cultured
Sertohi cells depended
partially
on the cell plating
density
and on the duration
of
the collection
period.
Importantly,
the Tf secretion
ratio
(apical/basal)
was less than 1.0 in cultures
in which fewer
than 2 x 106 cells were plated per well, even though
those
cultures
lacked
hydrodynamic
equilibration.
A significant
aspect of Sertoli cells cultured
at low cell density
is that the
tall columnar
shape of the cells was not maintained,
even
through
the Sertoli cells formed
a monolayer
by spreading
on the filters and contacted
one another
(Fig. 4). In these
flattened
monolayers,
the number
of tight junctions
may be
less and/or
the integrity
of the junctional
complex
may vary
significantly
from those of more polarized
Sertoli cells. Thus,
proteins
secreted
into the apical chamber
by more
squamous Sertoli
cell monolayers
may not encounter
a permeability
barrier
and diffuse freely into the basal compartment. Alternatively,
the intracellular
distribution
of secretory
organelles
of squamous
Sertoli cells may be inadequate
for
expression
of polarized
protein
secretion.
As Figure 4 shows,
the cytoplasm
was higher
at 2 X 106 cells/well
than at 2.4
x 106 cells/well
or 3.6 X 106 cells/well.
We think the reason for this is merely
a normal
variation
in this particular
section.
Therefore,
the electrical
resistance
probably
depends on the formation
of tight junctions,
not on the height
of the cytoplasm
of cell layer.
Another
important
culture
parameter
described
in the
present
report
that may account
for the equivocal
results
regarding
the polarized
secretion
of If by Sertohi cells is
the duration
of the collection
period
used to measure
Tf.
Polarized
If secretion
was not observed
when the collection period
was extended
to 48 h, even in cultures
plated
with cells at high cell density.
Although
a definitive
expla-
682
ONODA
of these results is not available
at this time, we wish
to offer three possibilities
that may partially
explain
them.
First, the prolonged
48-h collection
period
may deplete
nutrition and buffering
capacity
of the culture
media, leading
to impaired
Sertoli
cell function.
If culture
media
are replaced
frequently
(e.g. every 12 h for 2 days) by fresh media, Tf secretion
ratio (apical/basal)
is maintained
at a 1.52.0 level [4]. Under these conditions,
nutritional
effects for
maintaining
tight junctions
may be sufficient.
Second,
proteases that may compromise
the permeability
barrier
may
accumulate
in the medium
during
a 48-h incubation.
For
example,
Aihenberg
and Fritz [10] reported
that elevated
plasminogen
activator
levels were
associated
with a decreased
integrity
of the permeability
barrier
generated
by
Sertohi cells, and that the increase
in permeability
of the
barrier
in Sertohi cells could be prevented
by the addition
of various
protease
inhibitors.
Third,
Sertoli
cells in vitro
may express
the ability to secrete
If differentially
in an apical or basal direction
in response
to medium
composition.
Whereas
during
initial culture
periods
the Sertoli cells may
preferentially
secrete
If in an apical direction,
once a desired concentration
of Tf is reached
in the apical medium,
the cells may alter their preferential
Tf secretion
in a basal
direction.
Moreover,
we do not know at this time whether
Tf secretion
is intrinsically
bidirectional
as shown
in Figure
6,
or if the bidirectional
secretion
is a peculiar
feature
of the
culture
system
in vitro. Therefore,
the nature
of the intracellular
mechanisms
responsible
for the polarized
secretion of Tf remain
to be clarified.
In this regard,
the interaction
of Sertohi cells with other
cell types governing
Tf
secretion
cannot
be overlooked.
For example,
recent studies report that germ cells modify
Sertoli cell activity [15, 34]
and that peritubular
cells influence
If secretion
by Sertohi
cells [35].
However,
the polarized
secretion
of If by Sertohi cells
in vitro is probably
a function
of the cells’ ability to retain
their in vivo phenotypic
characteristics.
Thus, we examined
the density-dependent
morphological
appearance
of Sertohi
cells in vitro to determine
the role of this parameter
on If
secretion.
To this end, Sertoli
cells were
cultured
on a
semipermeable
basement
membrane
consisting
of several
extracellular
matrix components
and at cellular
concentrations, chosen
empirically,
that enhance
the morphological
appearance
of the cells so as to better
mimic their in vivo
structural
counterparts.
Under
these conditions,
vectorial
secretion
of If occurred
in a manner
consistent
with a paracrine function
of Sertohi
cells in the maintenance
and regulation
of spermatogenesis.
Tf has been shown
to mediate
the transcellular
transport
of iron in vitro [20, 23] and in
vivo [5] and has been immunolocalized
in developing
germ
cells present
in the adluminal
compartment
of the seminiferous
epithelium
[36]. Although
the critical
step(s)
of
spermatogenesis
regulation
by iron remains
unclear,
significantly,
Tf receptors
have been detected
and measured
nation
El
AL.
on germ
cells residing
Thus, by using
within the adluminal
compartment
our model in vitro bicameral
chamber system and controlling
the plating
density
of Sertoli cells,
further
analysis
of polarized
Tf secretion
should
provide
greater
insight
into iron-mediated
molecular
events
governing
the regulation
of spermatogenesis.
[36-38].
ACKNOWLEDGMENT
The authors
wish to thank Mr. Hai-Ntng
preparation
of the microscopic
sections.
for his technical
Dal
in the
assistance
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