BIOLOGY
OF
REPRODUCTION
19,
7 12-719
(1978)
Some Morphological
and Functional
of the Porcine S. STOKLOSOWA,
Characteristics
of Cells
Theca Interna
in Tissue Culture1
J. BAHR2’3
E. GREGORASZCZUK
Department
and
of Animal
Institute
Physiology,
of Zoology,
Jagiellonian
University,
Krakow,
Poland
and of Animal Science2, Genetics Laboratory,
Department Animal
University
of Illinois,
Urbana,
Illinois
61801
ABSTRACT Theca
interna
cycle. The occasionally
cells
were
were
follicles
from
large
porcine
theca interna layer was manually separated treated with hyaluronidase solution and
consisted of approximately cells. In one experiment, to
collected
compared.
lipid
visualize
70% theca cells collected Specific
droplets
in
interna from
cell the
types
follicles
from the trypsinized.
cells, 8% granulosa the granulosa layer were identified by:
cytoplasm;
2)
in the
theca
phase
1)
staining
of
with
the
Oil
(s’, 3/3-OH SH) enzyme and 3) quantitating estrogen production Nonsteroidogenic cells do not stain by any of the techniques applied. Theca interna cells can be distinguished from granulosa cells in suspension
the following 3) larger cell
following enzyme
1) more intense Theca interna cells
criteria:
size.
parameters: activity
1) more
and
4) increased
staining in culture
with ORO; 2) stronger can be differentiated
elongated
shape;
estrogen
production.
INTRODUCTION Bjersing ning using and
Later
ovaries
pure and granulosa
Nalbandov,
culture
No.
of
Fischer
Results granulosa
were
cells them
(Korenman,
conditions,
synthesize There interna hormone
cells 1971;
of the cow and cultured. showed that
(1970a)
granulosa to grow
the
first
from the in tissue
the
rat
and
Kahn,
1973)
of cells exhibit
steroid hormones. have been similar cells which synthesis.
and
previously
these adapt
(Nekola
April 28, 1978 February 22, 1977 by NIH PL 480
05-001-0, 3Reprint
a-nex requests
1972)
good reports
Research
of
chaotic
been
cultured
to culture
growth;
by
3) weaker
investigated
in short or in
Ryle et al., experiments
Therefore, interna cells
and
growth
prior
the
ex-
employing
term organ
incubation culture
1973; Rondell, (Channing
(Lacroix, (Channing, and
1974) or in Coudert,
1976).
and
develop
were isolated experiments well to tissue
also participate Theca interna
Accepted Received ‘Supported
1969b; in vivo
to
ovarian culture.
by
, 3/3-OH SH activity and from granulosa cells by the
more
plants either et al., 1974)
(1962) using pig ovaries and Chanmare (1966, 1969a), human (1969b)
monkey
isolate follicles
2) slower
0 (ORO)
Red
3/3-hydroxysteroid
i3’,
dehydrogenase
cells.
of the
cells and 23% nonsteroidogenic and the theca interna of the same
activity
measuring
follicular
externa, washed thoroughly, The resulting cell suspension
it was our from porcine
primary
cultures
At the same time differences between cells monal
and
in
culture criteria.
an using
aim to ovarian grown
attempt granulosa
isolate follicles
theca and
as monolayers. was and
morphological
made theca
to find interna and
hor-
theca
in ovarian tissue has
MATERIALS
AND
METHODS
Ovaries Ovaries of pigs were obtained from slaughterhouse animals and classified according to Akins and Morrissette (1968) and Channing and Ledwitz-Rigby (1975). Ovaries with large follicles (1.0-1.2 cm in diameter) in the foilicular phase of the cycle were selected. Most ovaries contained corpora albicantia, while approximately 10% had pale pink regressing corpora lutea and about 3% had no luteal structure.
Agreement
05-040-N. to J. Bahr.
712
THECA
Immediately ovaries
were
#{149}afterremoval placed
in cold
following antibiotic 0.2 mg streptomycin to 3 h usually elapsed and the establishment
Isolation
of
Theca
from
sterile
saline
INTERNA
the
CELLS
animals,
containing
the
concentrations: 240 U penicillin; and 20 IU mucostatin/mI. Two between the removal of ovaries of cultures.
Interna
Tissue
Ovaries were thoroughly rinsed 3 times in liter beakers containing sterile phosphate buffered saline (PBS) with decreasing doses of antibiotics (Channing et al., 1975). Then as each ovary was held with surgical forceps, follicles were cut and as many granulosa cells as possible were removed from the follicles using a platinum loop. Then using sharp scalpel blades and Iris scissors, the follicles were excised and placed in a Petri dish. The follicles were cut open and spread on the bottom of a Petri dish in such a way that the interior, i.e., granulosa layer, was turned upwards. The remaining granulosa cells were gently scraped off with a blunt spatula or a rubber-policeman and the cells were washed out with a vigorous stream of PBS using a cotton plugged capillary pipette. The most external layer of the granulosa cells is sometimes attached very strongly to the basement membrane. In such cases the tissue was rinsed in a solution of hyaluronidase in a concentration of 10 lU/mI of PBS. After this treatment granulosa cells detached more easily. The theca layers were then placed in a drop of saline under the dissecting microscope. The theca interna was manually separated from the underlying vascular theca externa. The theca interna pieces were then stirred vigorously in Medium 199 with a magnetic stirrer. This procedure was employed to avoid contamination with hyaluronidase which inhibits the growth of cell colonies. Next, tissue was transferred with fine forceps to a clean Petri dish containing fresh Medium 199 and was energetically pipetted many times with a capillary pipette with a large orifice. The medium was changed many times until no granulosa cell clusters were visible when checked under the dissecting microscope.
IN TISSUE
cells. Another (Fischer and ,
of
Cell
was Kahn,
713
submitted 1972)
3/3-hydroxysteroid
to
to a histochemical demonstrate activity
dehydrogenase
SD), an enzyme which sis in steroid producing
promotes cells.
(5,
progesterone Two ahiquots
test of 3/3-OH
synthe(0.1 ml
each) of theca interna cell suspension obtained after trypsinization were treated in the same manner. Simultaneously, granulosa cells from the same follicles were submitted to the same staining procedures to establish differences between theca and granulosa cells prior to culture. This procedure helped to establish the percentage of theca interna cells in the suspension prepared for culture and also to identify contamination with other cell types (Fig. 1). To distinguish theca interna cells from granulosa cells in culture the following experiment was performed. Granulosa cells and theca interna layers were collected from 80 preovulatory follicles. A cell suspension of theca interna was prepared in the manner described above. From 80 follicles, about 15 X 10’ granulosa cells/mi of the medium were obtained while from the thecae of the same, follicles only 3.5 X io cells/mI were recovered. The amount of cells collected was sufficient to set up 20 cultures of theca and 20 cultures of granulosa cells, maintaining cell concentrations as mentioned above. Cells were grown as separate monolayers simultaneously for 8-15 days. The medium from 4 cultures of granulosa cells and theca interna cells was collected every second day for steroid analysis. The remaining cultures were used for morphological investigation. Every other day, 1 or 2 cultures were terminated and monolayers were stained with May-Grunwald-Giemsa stain (Merchant et al., 1968) or ORO and examined histochemically for , 3/3-OH SH.
70
CRC (+)
w (I) +1
lx Preparation
CULTURE
Suspension
50
Q)
Isolated theca interna tissue that had been washed and cleaned was minced with scissors and exposed to routine gentle trypsinization with 0.25% trypsin (Difco) in PBS and was repeated 3 times for 10 mm at 37#{176}C. Following this treatment, the theca interna tissue was dispersed. The resulting cell suspension
contained
viable
cells
many
of which
were
ovate
Several divided sample 1959)
of Theca
pieces
Interna
of cleaned
into 2 samples was stained with to visualize lipid
0
30
ORO(-)
U)
a) 0
or
polygonal. Cells were tested for viability with trypan blue stain. Only about 10% of cells were damaged by trypsin. Much connective tissue from the stroma of the theca interns layer was left after trypsinization. The cell suspension was centrifuged after trypsinization. The pellet was then washed with Medium 199 and the cells were resuspended in culture medium.
Identification
C 0
Cells
theca interns tissue were prior to trypsinization. One Oil Red 0 (ORO) (Casselman, droplets within steroidogenic
ORO(+) I0
Theco cells
Nonsteroidogenic cells
Gronuloso cells
FIG. 1. Distribution of cells (%) in suspension obtained from theca interna tissue prior to culture. ORO (i-) represents cells stained with Oil Red 0; ORO (-) represents cells which did not stain with Oil Red 0. The ORO (+) cells, according to uptake of stain, were identified as either theca interna (70%) or granulosa cells (8%).
STOKLOSOWA
714
Medium
and
Culture
System
ET AL.
the
Medium 199 (Laboratory of Sera and Vaccines, Lublin, Poland) supplemented with 15% of calf serum was used. No quantitative measurements of LH concentration in this serum was made. In subsequent experiments, the amount of calf serum was reduced to 10 and 7% and no difference in cell growth or morphology was observed. No attempt was made to measure FSH in the serum. Progestin content in the serum measured by a Protein Binding Assay (Neill et al., 1967) was nondetectable. The estrogen measured by RIA (Hotchkiss et al., 1971) was 6-8 pg/mI. This amount was within blank values. Cells were grown as monolayers in Leighton tubes at 37#{176}C.Medium was changed every second or third day. Oxygenation of cultures after changing the medium was not essential for the growth of cells.
cell
us to types
(Fig.
cells
histochemically.
prior
to
within
the
cells
exhibit by
are
cells
of
more
rather
compact
colonies
morphology. of
tured
tory
ovarian
layer
follicle
of
from
the
the
pig
preovula-
consists
primar-
cells
,
after
thecal
tissue
(Fig.
tion
stain
313-OH
SH
these
cells
cell
culture.
layer
with
activity, at
any
to
while
granulosa
droplets
which
FIG. FIG. Fig. 3 granulosa granulosa
Cells
piece
of theca
Theca heavy
interns formazan
Granulosa SH. Activity
lipid
that
interna
cells.
prior
12
in
present
theca
the
and The
granu-
intensity
to
3j3-OH
but
11, of
shown
12).
cells
of
in
Table
of
culture,
thecal
number
tube
of
was
estrogen
trypsinization
produc-
from
1.
cells cells
the
cells. in
separate
It
is obvious
the
estrogen
predominates inoculated
into
1/5
stained
The
thecal
with
of
tremendous
cell
Oil
same
as
approximately
granulosa
SH,
weaker
estrogen
simultaneously
the
in
,
(Figs.
granulosa
cultures
the
granulosa
the
Red
0.
culture
on
Dark
areas
X 128.
prior
cells in deposits.
layer
of
decrease
of
cul-
a distinct
beginning
in
fewer
cells
cells from of the
content
have
surface
and
the
though
the
the
stain
and
results
are
at
culture
suspension
typical
ORO
culture.
exhibit
grown
even
to
cells
theca
the
4. Granulosa cells prior to and stained with ORO. X cells magnified 900 X shows than theca cells.
FIG. 5. contain
by
that
a
have
10 show
in
of
response
lipid
in
theca
interna
as
in
is
form
9 and
Cultured
preliminary
with
cells
isolated
of
11
SH
activity
monolayers
identify
whether
The
follicles
,
Figs.
3/3-OH
grow body
cells
cells
of
culture.
histochemical
packed
closer
of
to
or
of theca
3. Suspension
FIG. 6. 3/3-OH X 320.
a5,
A
exhibit
preparation
cells,
densely
appear
topography
also
the
trypsinization
droplets,
2.
possible
is
interna
are
FIG.
it
during
culture,
show
and
time
Theca
before
prior
ORO
cell
granulosa
5 days
to
while
selectively
cells
the
8
granulosa
interna
and
and
cells
prior stronger
cells
and
and
is
7
cells show
steroidogenic
the
cells in
ovoid and polygonal cells arranged in embedded in a stroma of connective After isolation and washing, the theca layer appears as a thin vascular sheet of Because
while
of this histochemical reaction in cultured is reversed when compared with the intensity
ily of groups tissue. interna
2).
and The
Figures
Figures
while
of
within
theca in size. difference
tissue
distribution
cells,
This
Theca
luteal
thecal activity
diameter,
manner while
losa
interna
culture.
elongated,
activity
theca
in
interna
chaotic
manner
contrary,
culture.
theca
cells
deposits
pm. in
when granules
Moreover, differ
pm
a 6 day
a more
the
formazan
6).
17-23
cell the
true
enzyme
suspension
also
in
On
10-14 are
cells in
in
is
formazan
intensive
5,
cells
same
few
heavy
(Figs.
granulosa
enabled
different into
Granulosa
show
very
cells
difference
The
cytoplasma.
expressed
the
1).
culture
maintained
RESULTS
This
tube
former
Analysis
4).
culture
latter
Estrogen secreted into the culture medium was measured by RIA (Flotchkiss et al., 1971) using antibody kindly donated by Dr. B. Caldwell, Yale University and l I-I] estradiol (New England Nuclear). The standard curve extended from 5-200 pg of cold estradiol (Sigma). This antibody cross reacts 40% with estrone and 6% with estriol.
The
3,
as a percentage, the suspension inoculated
testing
show Steroid
(Figs.
express, in the
to culture
stained
with
Oil
Red
0.
X 320.
culture. Cells were collected from the same 320. In the right hand upper corner, the more distinctly that ORO positive granules
suspension X 320.
submitted
the same follicles as cells enzyme is very weak. Note
to
the
histochemical
in Fig. 5 submitted to size difference between
test
follicles insert, are
for
,
as cells in a picture of fewer within
3/3-OH
the histochemical theca and granulosa
test
SH.
for cells.
THECA
INTERNA
CELLS
IN TISSUE
CULTURE
715
-.:
f3
:
-
.-
716
STOKLOSOWA
Day
4 is partially
caused
by the
fact
that
ET AL.
many
in vivo.
thecal cells have not attached to the glass and are lost during the medium change. In contrast granulosa
cells
The being
attach
growth investigated
of
to glass
more
both cell in current
is It
cells,
especially
when
and
maintaining
theca
cells
the
as
ratio
existing
the
the
the shape fibroblastic. nature
of
end
culture
(15-20
appear
in many
cells.
connected
of cultured
This
with
and
this
data
presented
interna dispersed
From
the
tissue and
that
that
morphology,
and
i,
3/3-OH
granulosa and it is possible prior
to
the
SH
cells
of
activity
culture.
The
enzyme
in
to culture and by the finding
preovulatory
rats
in proestrus
SH
for
the
period
that cells
difference
in
in cultured cells that granulosa
activity
of
immediately
the
in
LH
cells
the
FIG
Giemsa FIG.
A
7.
stain.
monolayer
8.
A
(Noworyta
of
(Bjersing, of this
theca
monolayer of granulosa typical shape of cells
FIG 9. Cultured
theca
cells stained
A corresponding
granulosa
FIG
10.
FIG
11. A X 200.
FIG.
rat
and
after
the
number
incubation
more
testosterone.
of tube,
estrogen
Channing
an
monkey
addition
the first data, it
was
cells (Table 1). Later on decreases which may a lack of the necessary
noticed
in
increase
thecal
of
LH
(unpub-
in
estrogen
fragments
to
when Furtherreported
thecal
cells
in
experiments.
From
glucose-6-phosphate
data
presented
that
days
our
dehydrogenase,
and alkaline phosphatases, cultured granulosa and
clude cells.
6
current
results (Stadnicka and Stoklosowa, we know that there are differences pattern of the activity of several enzymes as
acid rately
1967) enzyme
cells
times
secrediffer-
in
the
culture
here
cultured
stained
etc. theca
encourage
in sepacells. All us
to
con-
cells
were
theca
interna
with
the
May-Grunwald-
X 320.
Compare the stain. X320.
culture.
the
the
the
enzyme.
the pig activity
1/5 per
rat
such
in
enzyme
in estrogen cells. The
that
and Szoltys, 1975). Before time, granulosa cells show no detectable activity of the
Theca
luteinize
the
medium. (1977)
surge (Noworyta and after this histochemically
Szoltys, 1975) and in also show a very intense
7-13
by
previous 1976)
3/3-OH
,
not of
testosterone was added to the more, Fortune and Armstrong
gation
from
after
is
culture greatly enhanced the production of testosterone. All these factors are under investi-
and
removed
It
12).
though
the former of estrogen caused by
data)
synthesis
in
and of
granulosa
follicles
show
a short
different
are
(Figs. 5-12) both types
11,
cultured
i.e.,
lished
distribution
during the
were
precursors,
it is appar-
droplet
of
theca cells prior can be explained
follicular
experiment, lipid
theca cells to distinguish
and
activity
that
do
activity
differences and theca
even
cells
secreted by the amount possibly be
of pig ovary can be sepacultured as a monolayer.
comparative
ent
(Figs.
approximately
show
obtained
culture.
probably the
are also granulosa
is seen
cells. DISCUSSION
The
results
to
ence is very striking, especially during 2 days in culture. From our preliminary
degenera-
thecal
theca rated,
cells
reason,
weaker
There by
tion
phenomenon
aging
our
prior
follicles at the stage when this its highest activity in granulosa
theca
for
is much
days),
both types of cells becomes more Vacuoles and granules of unknown
is probably tion
of
to find exhibits
contrary, and
follicle.
Toward
with
in slaughterhouse animals. In such cases, in suspension reflect the situation in vivo. In culture, granulosa cells spontaneously luteinize (Channing, 1970b) and exhibit a stronger activity of , 3/3-OH SH which is involved in progesterone synthesis. On the
of
in
coincides suspensions
cells cells
appears from our observation that the growth of thecal cells is satisfactory, but to a certain extent, not as rich as the growth of granulosa granulosa
cell
difficult enzyme
readily.
types in culture experiments.
This
from
monolayer
12. A corresponding
of
theca
monolayer
cells obtained with that of with
ORO.
the same follicles Cells were stained
as were the cells in Fig. with May-Grunwald-Giemsa
7.
X 320.
cell culture cells
from theca.
stained showing
of granulosa
with
ORO.
activity cells.
X 200.
X 320.
of
the
,
3/3-OH
SH
after
5
days
in
THECA
INTERNA
CELLS
IN TISSUE
717
CULTURE
r.
4!
5. -I
a
;ji:4
I
4 a
I
-.
718
STOKLOSOWA
TABLE 1. Estrogen and 3.5 X iO theca
content interns
ET AL.
of the media of theca interns cells were cultured in separate
and granulosa tubes.
Estr ogen
(pg/b5
Days Celltype Theca
2 interna
Granulosa
cells
±
SEM of 4 cultures.
bMean
±
SEM
We
26.10 1316b 5.69a 671b
42.40
aMen
would
of 4 cultures,
like
incomplete
to emphasize
again
data
since
that
when
of
70% cells.
theca The
nonsteroidogenic staining.
as It
percentage culture
appears
interna remaining
of granulosa be overgrown
will
by
They
and
form
do
not
2.12a
18.56
1.32a
29.33
2.81a
11.46
1.61k
5.0
O.20
the
cells in a theca cell by the more numer-
overgrow
monohayers
usually stain
do very
Giemsa
stain
fainter.
In
interna
cells
the
as rapidly
when intensely
cultured with
while
the
another
as
fibroblasts
of theca
staining
cells
in which
subcultured
cells,
but
are
theca
interna
is
theca
(Stoklosowa
and
Szohtys, In Press), they recovered their hal morphology and secreted estrogen. additional evidence that these cells fibroblastic
cells
alone. Fibroblasts the May-Grunwald-
experiment,
were
theca
epitheThis is are not cells.
ACKNOWLEDGEMENTS We are greatly dov
for
of the
his
indebted to Professor interest, support to Dr. C. Channing
constant
manuscript;
suggestions
and
to
Clara and Mrs. clerical assistance.
Dr.
Jane
B. Grygon, Harris for
A. V. Nalban-
and
Mrs.
their
correction for
valuable
Alberta
Mc-
technical
and
REFERENCES Akins,
E.
L.
and
Morrissette,
M.
ovarian changes during estrous Am. J. Vet. Res. 29, 1953-1955.
C.
(1968).
cycle
Gross
of swine.
8
21.42
ous viable theca cells provided the mitotic rate of the two cell types is not markedly different. The 23% of the cells which did not stain and which probably are fibroblasts do not play a significant role at least until 12-15 days of culture.
h)
3.004
CR0 a small
such
cells
31.42
cells and 8% cells were
determined logical that
granulosa
6
establishing a cell culture of a tissue we want to adapt to an in vitro condition, it is critical to know what specific types of cells are inoculated into culture tube. In this experiment cultures consisted granulosa
cells/48
15 X i0
in culture
4
298.99 446.43
cells
cell cultures.
experiment
is in
progress.
Bjersing, L. (1962). Methods for isolating pig granuloss cell aggregates in amounts allowing biochemical investigation of steroid hormone synthesis in vitro. Acts Pathol. Microbiol. Scand. 55, 127128.
Bjersing,
L. (1967).
, 3/3- and activities in 2953 04. Casselman, W.G.B.
I-Iistochemical 17/3 hydroxysteroid porcine ovary.
demonstration
of
dehydrogenase
Histochemie.
10,
(1959). Histochemical Technique. Methuen et Co. LTD, London and John Wiley and Sons, Inc., NY. pp. 64-90. Channing, C. P. (1966). Progesterone biosynthesis by equine granulosa cells growing in tissue culture. Nature,
Channing, ovarian
C.
London 210, P. (1969a).
phology.
1266. Tissue
culture
cell types: Culture methods J. Endocrinol. 43, 403-414.
of
and
equine
mor-
C. P. (1969b). Steroidogenesis and morphology of human ovarian cell types in tissue culture. J. Endocrinol. 45, 297-308. Channing, C. P. (b970a). Effects of stage of the menstrual cycle and gonadotrophins on luteinization of Rhesus monkey granulosa cells in culture. Channing,
Endocrinology
87,
49-60.
Channing, C. P. (1970b). Effect of stage of the estrous cycle and gonadotrophins upon luteinization of porcine granulosa cells in culture. Endocrinology 87,
156-164.
Channing, C. P. and Coudert, S. (1976). Contribution of granulosa cells and follicular fluid to ovarian estrogen secretion in Rhesus monkey in vivo. Endocrinology 98, 590-597. Channing, C. P. and Ledwitz-Rigby, F. (1975). Methods for assessing hormone mediated differentiation of ovarian cells in culture and in short term incubations. In: S. P. Colowick and N. 0. Kaplan. Methods in Enzymology. XXXIX. Part D. (J. B. Hardman and B. O’Malley, eds.). Academic Press, New York, London. pp. 183-230. Fischer, T. V. and Kahn, R. H. (1972). Histochemical studies of rat ovarian follicular cells in vitro. In Vitro 7, 201-205. Fortune, J. E. and Armstrong, D. T. (1977). Androgen production by theca and granulosa isolated from proestrous
1341-1347.
rat
follicles.
Endocrinology
100,
THECA
INTERNA
CELLS
Hotchkiss, J., Knobil, E. and Atkinson, L. E. (1971). Radioiinmunoassay of estradiol in small volumes of peripheral plasma. Endocrinology 89, 177183. Korenman, S. G., Lolc, P. M., Beceiro, I., Sherman, B. M. and Granner, D. K. (1973). Long term culture of differentiated granulosa cells from individual bovine follicles. Endocrinology 93, 1423-1427. Lacroix, E., Eechaute, W. and Leusen, I. (1974). The biosynthesis of estrogens by cow follicles. Steroids 23, 337-356. Merchant, D. J., Kahn, R. H. and Murphy, W. H. (1968). Handbook of Cell and Organ Culture. Burgess Publishing Company. Neill, J., Johansson, E., Darts, J. and Knobil, E. (1967). Relationship between the plasma levels
IN TISSUE
CULTURE
Rondell, P. (1974). Role of steroid synthesis in the process of ovulation. Biol. Reprod. 10, 199-21 5. Ryle, M., Kent, J. and Boggis, J. (1973). Some observations on the theca and developing ovarian follicles. Ann. Biol. Anim. Bioch. Biophys. 13,
223-228. Stadnicka, A. and Stoklosowa, 5. (1976). Enzyme histochemistry of cultured ovarian cells. II. Histochemistry of porcine theca interna cells in tissue culture. Z. Mikrosk. -Anat. Forsch., Leipzig 90, 458-4-68. Stoklosowa, S. and Szoltys, M. Hormonal activity of cultured ovarian cells as measured by RIA. Proc. IlId Int. Symp. Immunol. in Reprod. September 1975. Varna, Bulgaria. In Press.
of luteimzing
hormone and progesterone during normal menstrual cycle. J. Chin. Endocrin. 1167-1173. Nekola, M. V. and Nalbandov, A. V. (1971). Morphological changes of rat follicular cells as influenced by oocytes. Biol. Reprod. 4, 154-160. Noworyta, B. and Szoltys, M. (1975). Dynamics of the , 3/3-hydroxysteroid dehydrogenase activity in rat follicular granulosa cells during proestrus. Bull. Acad. Pol. Sc. Serie des sciences biologiques. the 27,
Ci V. XXIII,
65-69.
719
RECOMMENDED
J. H. (1977).
Dorringron, The
Ovary.
Academic Lindner,
(L.
Pres,
REVIEWS Steroidogenesis
Zuckerman
and
in vitro. B. Weir,
In: eds.).
NY. pp. 359-412.
H. R., Amsterdam, A., Salomon, Y., Tsafriri, A., Minrod, A.,s Lamprecht, S. A., Zor, U and Koch, Y. (1977). Introovarian factors in ovulation: Determinants of folliculr response to gonadotrophins. J. Reprod. Fertil. Suppl. 25, 215-23 5.
OF
REPRODUCTION
19,
7 12-719
(1978)
Some Morphological
and Functional
of the Porcine S. STOKLOSOWA,
Characteristics
of Cells
Theca Interna
in Tissue Culture1
J. BAHR2’3
E. GREGORASZCZUK
Department
and
of Animal
Institute
Physiology,
of Zoology,
Jagiellonian
University,
Krakow,
Poland
and of Animal Science2, Genetics Laboratory,
Department Animal
University
of Illinois,
Urbana,
Illinois
61801
ABSTRACT Theca
interna
cycle. The occasionally
cells
were
were
follicles
from
large
porcine
theca interna layer was manually separated treated with hyaluronidase solution and
consisted of approximately cells. In one experiment, to
collected
compared.
lipid
visualize
70% theca cells collected Specific
droplets
in
interna from
cell the
types
follicles
from the trypsinized.
cells, 8% granulosa the granulosa layer were identified by:
cytoplasm;
2)
in the
theca
phase
1)
staining
of
with
the
Oil
(s’, 3/3-OH SH) enzyme and 3) quantitating estrogen production Nonsteroidogenic cells do not stain by any of the techniques applied. Theca interna cells can be distinguished from granulosa cells in suspension
the following 3) larger cell
following enzyme
1) more intense Theca interna cells
criteria:
size.
parameters: activity
1) more
and
4) increased
staining in culture
with ORO; 2) stronger can be differentiated
elongated
shape;
estrogen
production.
INTRODUCTION Bjersing ning using and
Later
ovaries
pure and granulosa
Nalbandov,
culture
No.
of
Fischer
Results granulosa
were
cells them
(Korenman,
conditions,
synthesize There interna hormone
cells 1971;
of the cow and cultured. showed that
(1970a)
granulosa to grow
the
first
from the in tissue
the
rat
and
Kahn,
1973)
of cells exhibit
steroid hormones. have been similar cells which synthesis.
and
previously
these adapt
(Nekola
April 28, 1978 February 22, 1977 by NIH PL 480
05-001-0, 3Reprint
a-nex requests
1972)
good reports
Research
of
chaotic
been
cultured
to culture
growth;
by
3) weaker
investigated
in short or in
Ryle et al., experiments
Therefore, interna cells
and
growth
prior
the
ex-
employing
term organ
incubation culture
1973; Rondell, (Channing
(Lacroix, (Channing, and
1974) or in Coudert,
1976).
and
develop
were isolated experiments well to tissue
also participate Theca interna
Accepted Received ‘Supported
1969b; in vivo
to
ovarian culture.
by
, 3/3-OH SH activity and from granulosa cells by the
more
plants either et al., 1974)
(1962) using pig ovaries and Chanmare (1966, 1969a), human (1969b)
monkey
isolate follicles
2) slower
0 (ORO)
Red
3/3-hydroxysteroid
i3’,
dehydrogenase
cells.
of the
cells and 23% nonsteroidogenic and the theca interna of the same
activity
measuring
follicular
externa, washed thoroughly, The resulting cell suspension
it was our from porcine
primary
cultures
At the same time differences between cells monal
and
in
culture criteria.
an using
aim to ovarian grown
attempt granulosa
isolate follicles
theca and
as monolayers. was and
morphological
made theca
to find interna and
hor-
theca
in ovarian tissue has
MATERIALS
AND
METHODS
Ovaries Ovaries of pigs were obtained from slaughterhouse animals and classified according to Akins and Morrissette (1968) and Channing and Ledwitz-Rigby (1975). Ovaries with large follicles (1.0-1.2 cm in diameter) in the foilicular phase of the cycle were selected. Most ovaries contained corpora albicantia, while approximately 10% had pale pink regressing corpora lutea and about 3% had no luteal structure.
Agreement
05-040-N. to J. Bahr.
712
THECA
Immediately ovaries
were
#{149}afterremoval placed
in cold
following antibiotic 0.2 mg streptomycin to 3 h usually elapsed and the establishment
Isolation
of
Theca
from
sterile
saline
INTERNA
the
CELLS
animals,
containing
the
concentrations: 240 U penicillin; and 20 IU mucostatin/mI. Two between the removal of ovaries of cultures.
Interna
Tissue
Ovaries were thoroughly rinsed 3 times in liter beakers containing sterile phosphate buffered saline (PBS) with decreasing doses of antibiotics (Channing et al., 1975). Then as each ovary was held with surgical forceps, follicles were cut and as many granulosa cells as possible were removed from the follicles using a platinum loop. Then using sharp scalpel blades and Iris scissors, the follicles were excised and placed in a Petri dish. The follicles were cut open and spread on the bottom of a Petri dish in such a way that the interior, i.e., granulosa layer, was turned upwards. The remaining granulosa cells were gently scraped off with a blunt spatula or a rubber-policeman and the cells were washed out with a vigorous stream of PBS using a cotton plugged capillary pipette. The most external layer of the granulosa cells is sometimes attached very strongly to the basement membrane. In such cases the tissue was rinsed in a solution of hyaluronidase in a concentration of 10 lU/mI of PBS. After this treatment granulosa cells detached more easily. The theca layers were then placed in a drop of saline under the dissecting microscope. The theca interna was manually separated from the underlying vascular theca externa. The theca interna pieces were then stirred vigorously in Medium 199 with a magnetic stirrer. This procedure was employed to avoid contamination with hyaluronidase which inhibits the growth of cell colonies. Next, tissue was transferred with fine forceps to a clean Petri dish containing fresh Medium 199 and was energetically pipetted many times with a capillary pipette with a large orifice. The medium was changed many times until no granulosa cell clusters were visible when checked under the dissecting microscope.
IN TISSUE
cells. Another (Fischer and ,
of
Cell
was Kahn,
713
submitted 1972)
3/3-hydroxysteroid
to
to a histochemical demonstrate activity
dehydrogenase
SD), an enzyme which sis in steroid producing
promotes cells.
(5,
progesterone Two ahiquots
test of 3/3-OH
synthe(0.1 ml
each) of theca interna cell suspension obtained after trypsinization were treated in the same manner. Simultaneously, granulosa cells from the same follicles were submitted to the same staining procedures to establish differences between theca and granulosa cells prior to culture. This procedure helped to establish the percentage of theca interna cells in the suspension prepared for culture and also to identify contamination with other cell types (Fig. 1). To distinguish theca interna cells from granulosa cells in culture the following experiment was performed. Granulosa cells and theca interna layers were collected from 80 preovulatory follicles. A cell suspension of theca interna was prepared in the manner described above. From 80 follicles, about 15 X 10’ granulosa cells/mi of the medium were obtained while from the thecae of the same, follicles only 3.5 X io cells/mI were recovered. The amount of cells collected was sufficient to set up 20 cultures of theca and 20 cultures of granulosa cells, maintaining cell concentrations as mentioned above. Cells were grown as separate monolayers simultaneously for 8-15 days. The medium from 4 cultures of granulosa cells and theca interna cells was collected every second day for steroid analysis. The remaining cultures were used for morphological investigation. Every other day, 1 or 2 cultures were terminated and monolayers were stained with May-Grunwald-Giemsa stain (Merchant et al., 1968) or ORO and examined histochemically for , 3/3-OH SH.
70
CRC (+)
w (I) +1
lx Preparation
CULTURE
Suspension
50
Q)
Isolated theca interna tissue that had been washed and cleaned was minced with scissors and exposed to routine gentle trypsinization with 0.25% trypsin (Difco) in PBS and was repeated 3 times for 10 mm at 37#{176}C. Following this treatment, the theca interna tissue was dispersed. The resulting cell suspension
contained
viable
cells
many
of which
were
ovate
Several divided sample 1959)
of Theca
pieces
Interna
of cleaned
into 2 samples was stained with to visualize lipid
0
30
ORO(-)
U)
a) 0
or
polygonal. Cells were tested for viability with trypan blue stain. Only about 10% of cells were damaged by trypsin. Much connective tissue from the stroma of the theca interns layer was left after trypsinization. The cell suspension was centrifuged after trypsinization. The pellet was then washed with Medium 199 and the cells were resuspended in culture medium.
Identification
C 0
Cells
theca interns tissue were prior to trypsinization. One Oil Red 0 (ORO) (Casselman, droplets within steroidogenic
ORO(+) I0
Theco cells
Nonsteroidogenic cells
Gronuloso cells
FIG. 1. Distribution of cells (%) in suspension obtained from theca interna tissue prior to culture. ORO (i-) represents cells stained with Oil Red 0; ORO (-) represents cells which did not stain with Oil Red 0. The ORO (+) cells, according to uptake of stain, were identified as either theca interna (70%) or granulosa cells (8%).
STOKLOSOWA
714
Medium
and
Culture
System
ET AL.
the
Medium 199 (Laboratory of Sera and Vaccines, Lublin, Poland) supplemented with 15% of calf serum was used. No quantitative measurements of LH concentration in this serum was made. In subsequent experiments, the amount of calf serum was reduced to 10 and 7% and no difference in cell growth or morphology was observed. No attempt was made to measure FSH in the serum. Progestin content in the serum measured by a Protein Binding Assay (Neill et al., 1967) was nondetectable. The estrogen measured by RIA (Hotchkiss et al., 1971) was 6-8 pg/mI. This amount was within blank values. Cells were grown as monolayers in Leighton tubes at 37#{176}C.Medium was changed every second or third day. Oxygenation of cultures after changing the medium was not essential for the growth of cells.
cell
us to types
(Fig.
cells
histochemically.
prior
to
within
the
cells
exhibit by
are
cells
of
more
rather
compact
colonies
morphology. of
tured
tory
ovarian
layer
follicle
of
from
the
the
pig
preovula-
consists
primar-
cells
,
after
thecal
tissue
(Fig.
tion
stain
313-OH
SH
these
cells
cell
culture.
layer
with
activity, at
any
to
while
granulosa
droplets
which
FIG. FIG. Fig. 3 granulosa granulosa
Cells
piece
of theca
Theca heavy
interns formazan
Granulosa SH. Activity
lipid
that
interna
cells.
prior
12
in
present
theca
the
and The
granu-
intensity
to
3j3-OH
but
11, of
shown
12).
cells
of
in
Table
of
culture,
thecal
number
tube
of
was
estrogen
trypsinization
produc-
from
1.
cells cells
the
cells. in
separate
It
is obvious
the
estrogen
predominates inoculated
into
1/5
stained
The
thecal
with
of
tremendous
cell
Oil
same
as
approximately
granulosa
SH,
weaker
estrogen
simultaneously
the
in
,
(Figs.
granulosa
cultures
the
granulosa
the
Red
0.
culture
on
Dark
areas
X 128.
prior
cells in deposits.
layer
of
decrease
of
cul-
a distinct
beginning
in
fewer
cells
cells from of the
content
have
surface
and
the
though
the
the
stain
and
results
are
at
culture
suspension
typical
ORO
culture.
exhibit
grown
even
to
cells
theca
the
4. Granulosa cells prior to and stained with ORO. X cells magnified 900 X shows than theca cells.
FIG. 5. contain
by
that
a
have
10 show
in
of
response
lipid
in
theca
interna
as
in
is
form
9 and
Cultured
preliminary
with
cells
isolated
of
11
SH
activity
monolayers
identify
whether
The
follicles
,
Figs.
3/3-OH
grow body
cells
cells
of
culture.
histochemical
packed
closer
of
to
or
of theca
3. Suspension
FIG. 6. 3/3-OH X 320.
a5,
A
exhibit
preparation
cells,
densely
appear
topography
also
the
trypsinization
droplets,
2.
possible
is
interna
are
FIG.
it
during
culture,
show
and
time
Theca
before
prior
ORO
cell
granulosa
5 days
to
while
selectively
cells
the
8
granulosa
interna
and
and
cells
prior stronger
cells
and
and
is
7
cells show
steroidogenic
the
cells in
ovoid and polygonal cells arranged in embedded in a stroma of connective After isolation and washing, the theca layer appears as a thin vascular sheet of Because
while
of this histochemical reaction in cultured is reversed when compared with the intensity
ily of groups tissue. interna
2).
and The
Figures
Figures
while
of
within
theca in size. difference
tissue
distribution
cells,
This
Theca
luteal
thecal activity
diameter,
manner while
losa
interna
culture.
elongated,
activity
theca
in
interna
chaotic
manner
contrary,
culture.
theca
cells
deposits
pm. in
when granules
Moreover, differ
pm
a 6 day
a more
the
formazan
6).
17-23
cell the
true
enzyme
suspension
also
in
On
10-14 are
cells in
in
is
formazan
intensive
5,
cells
same
few
heavy
(Figs.
granulosa
enabled
different into
Granulosa
show
very
cells
difference
The
cytoplasma.
expressed
the
1).
culture
maintained
RESULTS
This
tube
former
Analysis
4).
culture
latter
Estrogen secreted into the culture medium was measured by RIA (Flotchkiss et al., 1971) using antibody kindly donated by Dr. B. Caldwell, Yale University and l I-I] estradiol (New England Nuclear). The standard curve extended from 5-200 pg of cold estradiol (Sigma). This antibody cross reacts 40% with estrone and 6% with estriol.
The
3,
as a percentage, the suspension inoculated
testing
show Steroid
(Figs.
express, in the
to culture
stained
with
Oil
Red
0.
X 320.
culture. Cells were collected from the same 320. In the right hand upper corner, the more distinctly that ORO positive granules
suspension X 320.
submitted
the same follicles as cells enzyme is very weak. Note
to
the
histochemical
in Fig. 5 submitted to size difference between
test
follicles insert, are
for
,
as cells in a picture of fewer within
3/3-OH
the histochemical theca and granulosa
test
SH.
for cells.
THECA
INTERNA
CELLS
IN TISSUE
CULTURE
715
-.:
f3
:
-
.-
716
STOKLOSOWA
Day
4 is partially
caused
by the
fact
that
ET AL.
many
in vivo.
thecal cells have not attached to the glass and are lost during the medium change. In contrast granulosa
cells
The being
attach
growth investigated
of
to glass
more
both cell in current
is It
cells,
especially
when
and
maintaining
theca
cells
the
as
ratio
existing
the
the
the shape fibroblastic. nature
of
end
culture
(15-20
appear
in many
cells.
connected
of cultured
This
with
and
this
data
presented
interna dispersed
From
the
tissue and
that
that
morphology,
and
i,
3/3-OH
granulosa and it is possible prior
to
the
SH
cells
of
activity
culture.
The
enzyme
in
to culture and by the finding
preovulatory
rats
in proestrus
SH
for
the
period
that cells
difference
in
in cultured cells that granulosa
activity
of
immediately
the
in
LH
cells
the
FIG
Giemsa FIG.
A
7.
stain.
monolayer
8.
A
(Noworyta
of
(Bjersing, of this
theca
monolayer of granulosa typical shape of cells
FIG 9. Cultured
theca
cells stained
A corresponding
granulosa
FIG
10.
FIG
11. A X 200.
FIG.
rat
and
after
the
number
incubation
more
testosterone.
of tube,
estrogen
Channing
an
monkey
addition
the first data, it
was
cells (Table 1). Later on decreases which may a lack of the necessary
noticed
in
increase
thecal
of
LH
(unpub-
in
estrogen
fragments
to
when Furtherreported
thecal
cells
in
experiments.
From
glucose-6-phosphate
data
presented
that
days
our
dehydrogenase,
and alkaline phosphatases, cultured granulosa and
clude cells.
6
current
results (Stadnicka and Stoklosowa, we know that there are differences pattern of the activity of several enzymes as
acid rately
1967) enzyme
cells
times
secrediffer-
in
the
culture
here
cultured
stained
etc. theca
encourage
in sepacells. All us
to
con-
cells
were
theca
interna
with
the
May-Grunwald-
X 320.
Compare the stain. X320.
culture.
the
the
the
enzyme.
the pig activity
1/5 per
rat
such
in
enzyme
in estrogen cells. The
that
and Szoltys, 1975). Before time, granulosa cells show no detectable activity of the
Theca
luteinize
the
medium. (1977)
surge (Noworyta and after this histochemically
Szoltys, 1975) and in also show a very intense
7-13
by
previous 1976)
3/3-OH
,
not of
testosterone was added to the more, Fortune and Armstrong
gation
from
after
is
culture greatly enhanced the production of testosterone. All these factors are under investi-
and
removed
It
12).
though
the former of estrogen caused by
data)
synthesis
in
and of
granulosa
follicles
show
a short
different
are
(Figs. 5-12) both types
11,
cultured
i.e.,
lished
distribution
during the
were
precursors,
it is appar-
droplet
of
theca cells prior can be explained
follicular
experiment, lipid
theca cells to distinguish
and
activity
that
do
activity
differences and theca
even
cells
secreted by the amount possibly be
of pig ovary can be sepacultured as a monolayer.
comparative
ent
(Figs.
approximately
show
obtained
culture.
probably the
are also granulosa
is seen
cells. DISCUSSION
The
results
to
ence is very striking, especially during 2 days in culture. From our preliminary
degenera-
thecal
theca rated,
cells
reason,
weaker
There by
tion
phenomenon
aging
our
prior
follicles at the stage when this its highest activity in granulosa
theca
for
is much
days),
both types of cells becomes more Vacuoles and granules of unknown
is probably tion
of
to find exhibits
contrary, and
follicle.
Toward
with
in slaughterhouse animals. In such cases, in suspension reflect the situation in vivo. In culture, granulosa cells spontaneously luteinize (Channing, 1970b) and exhibit a stronger activity of , 3/3-OH SH which is involved in progesterone synthesis. On the
of
in
coincides suspensions
cells cells
appears from our observation that the growth of thecal cells is satisfactory, but to a certain extent, not as rich as the growth of granulosa granulosa
cell
difficult enzyme
readily.
types in culture experiments.
This
from
monolayer
12. A corresponding
of
theca
monolayer
cells obtained with that of with
ORO.
the same follicles Cells were stained
as were the cells in Fig. with May-Grunwald-Giemsa
7.
X 320.
cell culture cells
from theca.
stained showing
of granulosa
with
ORO.
activity cells.
X 200.
X 320.
of
the
,
3/3-OH
SH
after
5
days
in
THECA
INTERNA
CELLS
IN TISSUE
717
CULTURE
r.
4!
5. -I
a
;ji:4
I
4 a
I
-.
718
STOKLOSOWA
TABLE 1. Estrogen and 3.5 X iO theca
content interns
ET AL.
of the media of theca interns cells were cultured in separate
and granulosa tubes.
Estr ogen
(pg/b5
Days Celltype Theca
2 interna
Granulosa
cells
±
SEM of 4 cultures.
bMean
±
SEM
We
26.10 1316b 5.69a 671b
42.40
aMen
would
of 4 cultures,
like
incomplete
to emphasize
again
data
since
that
when
of
70% cells.
theca The
nonsteroidogenic staining.
as It
percentage culture
appears
interna remaining
of granulosa be overgrown
will
by
They
and
form
do
not
2.12a
18.56
1.32a
29.33
2.81a
11.46
1.61k
5.0
O.20
the
cells in a theca cell by the more numer-
overgrow
monohayers
usually stain
do very
Giemsa
stain
fainter.
In
interna
cells
the
as rapidly
when intensely
cultured with
while
the
another
as
fibroblasts
of theca
staining
cells
in which
subcultured
cells,
but
are
theca
interna
is
theca
(Stoklosowa
and
Szohtys, In Press), they recovered their hal morphology and secreted estrogen. additional evidence that these cells fibroblastic
cells
alone. Fibroblasts the May-Grunwald-
experiment,
were
theca
epitheThis is are not cells.
ACKNOWLEDGEMENTS We are greatly dov
for
of the
his
indebted to Professor interest, support to Dr. C. Channing
constant
manuscript;
suggestions
and
to
Clara and Mrs. clerical assistance.
Dr.
Jane
B. Grygon, Harris for
A. V. Nalban-
and
Mrs.
their
correction for
valuable
Alberta
Mc-
technical
and
REFERENCES Akins,
E.
L.
and
Morrissette,
M.
ovarian changes during estrous Am. J. Vet. Res. 29, 1953-1955.
C.
(1968).
cycle
Gross
of swine.
8
21.42
ous viable theca cells provided the mitotic rate of the two cell types is not markedly different. The 23% of the cells which did not stain and which probably are fibroblasts do not play a significant role at least until 12-15 days of culture.
h)
3.004
CR0 a small
such
cells
31.42
cells and 8% cells were
determined logical that
granulosa
6
establishing a cell culture of a tissue we want to adapt to an in vitro condition, it is critical to know what specific types of cells are inoculated into culture tube. In this experiment cultures consisted granulosa
cells/48
15 X i0
in culture
4
298.99 446.43
cells
cell cultures.
experiment
is in
progress.
Bjersing, L. (1962). Methods for isolating pig granuloss cell aggregates in amounts allowing biochemical investigation of steroid hormone synthesis in vitro. Acts Pathol. Microbiol. Scand. 55, 127128.
Bjersing,
L. (1967).
, 3/3- and activities in 2953 04. Casselman, W.G.B.
I-Iistochemical 17/3 hydroxysteroid porcine ovary.
demonstration
of
dehydrogenase
Histochemie.
10,
(1959). Histochemical Technique. Methuen et Co. LTD, London and John Wiley and Sons, Inc., NY. pp. 64-90. Channing, C. P. (1966). Progesterone biosynthesis by equine granulosa cells growing in tissue culture. Nature,
Channing, ovarian
C.
London 210, P. (1969a).
phology.
1266. Tissue
culture
cell types: Culture methods J. Endocrinol. 43, 403-414.
of
and
equine
mor-
C. P. (1969b). Steroidogenesis and morphology of human ovarian cell types in tissue culture. J. Endocrinol. 45, 297-308. Channing, C. P. (b970a). Effects of stage of the menstrual cycle and gonadotrophins on luteinization of Rhesus monkey granulosa cells in culture. Channing,
Endocrinology
87,
49-60.
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