and cytochalasin. B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For exam- ple, granulosa cell progesterone production.
BIOLOGY
OF
38,
REPRODUCTION
The
100-108
(1988)
Cytoskeleton
and Rat Granulosa
Possible
Involvement
Cell Steroidogenesis:
of Microtubules
and Microfilaments1 JACQUELINE
A. CARNEGIE3
Reproductive
Biology and
Unit,3
Department
and
BENJAMIN
Department
of
of Physiology,4
K. TSANG2’3’4’5
Obstetrics
and
University
of
Gynecology
Ottawa
and
The
Loeb
Institute Ottawa
Ottawa,
for
Medical
Civic
Hospital
Ontario,
Canada
Research5 KJY4E9
ABSTRACT The
monitoring (0-2.0 the
the pg/mI)
effects on cellular
cytochalasins
of
microtubule
or
cell
morphology
increased
(2Oca-OH-progesterone) was observed at 4-250 fluenced
both microtubules of colchicine (0-250
of
participation
and microfilaments in granulosa cell steroidogenesis was assessed pM) and/or cytochalasin B (0-10 iig/ml) or dihydrocytochalasin
and production
granulosa
cell
in a dose-dependent pM colchicine and
microfilament
spreading.
cells
for
stimulated
basal
24 h in medium
24 h of culture. and
The largest increase 4ug/ml cytochalasin. that
cultured
during
of progesterone
manner. at 2-10
polymerization
Whereas
of progestins
production
of
Both
progestin became
colchicine
and
20a-hydroxy-pregn-4-en-3-one
in steroidogenesis Those concentrations
alone
by B
(about 2- to 3-fold) of the inhibitors
production very
also
flatten-ed
markedly
with
in-
numerous
cytoplasmic extensions, those cultured with colchicine (0.2-250 $1) or cytochalasin (0.4-2 pg/mI) were much less spread and pro gessively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions
in mean
(2 pg/ml
cytochalasin
cytochalasin ple, granulosa chalasin a greater growth
contour
index
B), or 4.1
values ±
0.1
from
5.7
(2 pg/mI
B exhibited a greater steroidogenic cell progesterone production was
B, but extent surface
5.5-fold if both was
by 4 pM coichicine cytoskeleton-perturbing
reduced
by
60-70%
plus by
±
0.1
(control)
to 3.9
dihydrocytochalasin response stimulated
B).
than that elicited almost 2-fold
2 pg/mI cytochalasin agents were present;
4 pM
coichicine
± 0.1
Cultures
(250
pM
colchicine),
containing
both
by either inhibitor by 4 pM colchicine
2 /1g/ml
cytochalasin
±
0.1 and
alone. For examor 2 j.zg/mI cyto-
B. Similarly, cell spreading the mean area occupied by
or by
4.2
colchicine
B but
was reduced to the cells on the by
90%
in the
presence of the two. These findings suggest that both microtubules and microfilaments are important in granulosa cell steroidogenesis. We propose that the two cytoskeletal components function to facilitate the movement of cholesterol from lipid droplets to mitochondria, possibly through an effect on subcellular organelle distribution by altering the morphology of the granulosa cells.
INTRODUCTION
Steroidogenesis a number of example, substrate
is a biosynthetic distinct intracellular in the form of
dehydrogenase considerable intermediates
process involving locales. For cholesterol esters
we
have
are microsomal enzymes. intracellular transport of during steroid hormone proposed
is stored within lipid droplets, the cholesterol sidechain cleavage enzyme system is found on the inner membranes of mitochondria, and both the 313-hydro-
gonadotropic demonstrated
xystero
duction by microtubule-tubulin
id
dehydrogenase
and
mone
20a -hydroxysteroid
1987b). recognized Accepted July 29, 1987. Received May 19, 1987. ‘This work was supported Resesrch Council of Canada Ottawa Medical Foundation. 2 Reprint requests.
by grants (MT-7793)
(to and
B.K.T.) from the from the Kiwanis
a
regulation inhibition
that perturb equilibrium
However, cellular as integrated
cell
be
in and
progestin
the cytoplasmic (Carnegie et
cytoskeletons networks
including as well
cytoplasmic should
microtubules
this process follicle-stimulating
granulosa
agents
changes in the these constituents 100
for of of
(FSH)-stimulated
several components, intermediate filaments
Medical Club of
role
Due to the substrate and biosynthesis,
must consisting
microfilaments as microtubules,
distribution evaluated
of with
the have horproal., be of and and
one regard
of to
MICROTUBULES,
the
effect(s)
it may
wick and Porter, in addition to 1977; that
Sawyer
et al.,
gonadotropin tion in vitro 1981;
1979),
are hormone
(hCG) (Silavin
that
Gwynne
of others
et al., (Gemmell
it has
important (LH)
and
remain
unQlear
1982;
effects
of on
(Azhar
and
of the
steroidogenesis that inhibit (colchicine) dihydrocytochalasin gether,
on
the
culture.
levels
cellular filament Lin
by
et
al.,
1978),
in
has been transport
investigated
during
is known
as well as to (Tannenbaum
morphology
(Lin
shown and et al.,
to
and
the
24
h of
inhibit
block microet al., 1977;
this
to have little influence
also
and/or
toneal
follicular development Sprague-Dawley rats
injection
dotropin day of assured
on the morning birth). By using that
reached
the
ovaries Fortune
were and
which
of 4 IU
were
the
majority
antral
stage
harvested Armstrong, released
by
pregnant
cytochalasin
of Day 28 this treatment, of the of
Eagle’s
later The
gona-
1 (Day we
0 were
would
have
development
2 days 1977). into
follicles
when
the
(Day 30 ± 1; granulosa cells,
minimum
essential
medium (MEM; G IBCO Laboratories, Mississauga, Ontario) by follicle puncture and collected by centrifugation (180 X g, 10 mm), were treated sequentially with trypsin and DNase (Farookhi, 1982) to increase the
percentage
value of 20-30% this treatment cell suspension
of viability
of the
cells
B (0-10
B
(0-2
pg/mi).
directly
in
MEM.
The
sulfoxide
solvent
from
an initial
to a final level of 90-95%. Briefly, involved incubation of the granulosa (106 cells/ml) at 37#{176}Cwith trypsin
The
for
and
-one
or dihydrocytowas
final
added
treatment
at
the
of
progesterone
end
of
culture and
With
by al.,
exception
estrogens, both the
assays, of variation
perturbing
20a-
(20a-OH-progesterone)
the
studies
was
the
cross-reactivity with 20a -OH-progesterone, serum to 20cr-OH-progesterone cross-reacts
coefficients
of
cultures
and
20a-OH-progesterone
in
concentration
(Orczyk et The antiserum characterized
1979).
dissolved
were
specific radioimmunoassays after extraction with ether. gesterone has been previously Armstrong,
then
air in MEM containcolchicine (0-250 pM)
the
collected
pg/ml;
were
if they were to be at 36#{176}C under an
pg/mi)
determination
hydroxy-pregn4-en-3
cells
cytochaiasins
control
was
period
1(25
Coichicine
so that
in
0.2%. Medium
Chemical
DNase
95%
of
assay
serum ±
and
or no effect only cellular
was stimulated in a single intraperimare’s
5 mm.
0.1% with other progestins, (Morley et al., 1987). For
METHODS
(Sigma
with
for
chalasin
skeleton Ovarian immature
Co.)
Louis, MO) for digestion with
inhibitor
incubation
of 5% CO2 concentrations
respectively. For those
AND
trypsin
cytochalasin
1978).
MATERIALS
Co., St. enzymatic
for 24 h (on coverslips later using microscopy)
atmosphere ing various
this cell
B was as
to
Gwynne
produced
B
experiments,
derivative on sugar
al.,
bean
Chemical
dimethyl
dihydrocytochalasin
these
et
Chemical of the
finally,
cultured examined
influence of agents of microtubules (cytochalasin B, separately and tocells
soy and
Sigma
producMenon,
in granulosa
these
cytochalasin
uptake of glucose, polymerization
included
stimulation chorionic
1984). further
of progestins
assumed Since
excess
101
STEROIDOGENESIS
pg/mi; Sigma mm, cessation
Co.),
proposed
1981;
cytoskeleton
1
OVARIAN
been
Silavin
Menon,
by assessing the the polymerization or microfilaments B), both
configuration
(50
inhibitors of microbasal steroidogenesis
Condon, 1982; Silavin et al., In the current study, we have involvement
(Wolose-
in the or human
Condon,
although the polymerization
the
also
AND
1983). Indeed, and Stacy,
of ovarian progesterone et al., 1980; Azhar and
1984), filament and
on
1976; Herman microtubules
microfilaments luteinizing
by
have
MICROFILAMENTS
agents
on
of
8.7%
the less
antithan
and androgens progesterone and
the
intra-
and
were
