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Endocrinology
Copyright © 1998 by The Endocrine Society
Vol. 139, No. 4
Printed in U.S.A.
Establishment and Characterization of a Simian Virus
40-Transformed Temperature-Sensitive Rat Luteal Cell
Line*
N. SUGINO, M. ZILBERSTEIN, R. K. SRIVASTAVA, C. M. TELLERIA,
S. E. NELSON, M. RISK, J. Y. CHOU, AND G. GIBORI†
Department of Physiology and Biophysics (N.S., M.Z., R.K.S., C.M.T., S.E.N., M.R., G.G.) University of
Illinois at Chicago, Chicago, Illinois 60612-7342; Faulkner Centre for Reproductive Medicine (M.Z.,
R.K.S.), Boston, Massachusetts 02130; and Heritable Disorders Branch (J.Y.C.), National Institute of
Child Health and Human Development, Bethesda, Maryland 20892
ABSTRACT
The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the
possibility for the study of gene regulation in luteal cells. The goal of
this study was to develop a cell line to serve as a model by which to
study the expression and regulation of various genes specific to luteal
cells. We attempted to develop a luteal cell line by transformation of
large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive
mutation required for the maintenance of cell transformation. We
report here the successful establishment of such a cell line, designated
GG-CL cells. Large luteal cells were purified to homogeneity by flow
cytometry from corpora lutea of day 14 pregnant rats, cultured for
24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C)
until colonies were identified. Several colonies of transformed cells
were isolated and passaged. They multiplied at 33 C and formed
multilayers. At the nonpermissive temperature (40 C), cells reverted
to the normal differentiated phenotype similar to the primary luteal
cells in culture. To determine whether GG-CL cells express the genes
found in normal luteal cells, messenger RNA (mRNA) expression was
T
HE corpus luteum plays a pivotal role in the maintenance of pregnancy in the rat. During the cycle, the cells
of preovulatory follicles undergo rapid morphological and
functional changes to become luteinized. A well orchestrated
differentiation process in luteal cells underlies this marked
transformation. Recent studies in our laboratory identified a
variety of luteal cell-specific genes implicated in corpus luteum development and function. Most of the information to
date, however, has been derived from primary cell culture.
While this model is well characterized, the eventual yield is
very small and prohibitive to the design of large-scale endeavors necessary for the study of gene expression and regulation. Therefore, we have attempted to establish a temperature-sensitive cell line derived from the large luteal cells,
which combines abundance of cells with a constitutive moReceived October 29, 1997.
Address all correspondence and requests for reprints to: Dr. Geula
Gibori, Department of Physiology and Biophysics (M/C 901), University
of Illinois at Chicago, 901 South Wolcott Avenue, Chicago, Illinois
60612-7342.
* This work was supported by NIH Grants HD-11119 (to G.G.) and
FIC1F05TW05241 (to C.M.T.).
† Recipient of an NIH Merit Award (HD-11119).
examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for
interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-b (ER-b) and the orphan nuclear receptor nur 77.
No receptors for ER-a, progesterone, LH, or PRL could be detected.
This cell line also expressed 20a-hydroxysteroid dehydrogenase (20aHSD), but not cholesterol side-chain cleavage cytochrome P450
(P450scc), 3b-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL
receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected
with the PRL receptor, they responded to PRL with a marked inhibition in 20a-HSD mRNA expression. In addition, estradiol enhanced
ER-b expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the
orphan receptor nur 77. In summary, a temperature-sensitive cell line
was successfully established from the large luteal cells of rat corpora
lutea. These cells express key genes encoding enzymes and receptors
inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP. (Endocrinology 139: 1936 –
1942, 1998)
dality of temperature-driven differentiation (1). The established cell line also allows the stable introduction of wildtype genes where they were lost during the process of
transformation. We report here the establishment of a temperature-sensitive luteal cell line designated GG-CL that
does not express the enzymes involved in steroidogenesis
but retained many of the cell-specific elements encountered
in the primary cell of origin. They not only express estrogen
receptor-b (ER-b), glucocorticoid receptor (GR), interleukin-6 (IL-6) receptor, 20a-hydroxysteroid dehydrogenase
(20a-HSD), Janus kinase (JAK2), and signal transducer and
activator of transcription (Stat5), and the orphan nuclear
receptor nur 77, but also respond to estrogen with an enhanced expression of ER-b, to cAMP with a rapid induction
of nur 77, and to progesterone and glucocorticoid with an
inhibition in 20a-HSD (2). Since GG-CL cells are devoid of
PRL receptor, which is the focus of many ongoing studies in
our laboratory, we have successfully transfected this receptor
into these cells and showed that they also respond to PRL
with an inhibition in 20a-HSD. The data in this report confirm many of our previous observations in primary luteal cell
culture and attest to the great potential of the temperature-
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RAT LUTEAL CELL LINE
1937
TABLE 1. Oligonucleotide primers for RT-PCR
Predicted
size (bp)
Oligonucleotide primers for RT-PCR
ER-a
ER-b
PR
GR
PRL-RL
LH-R
IL-6R
20a-HSD
P450 scc
3b-HSD
P450 arom
JAK2
Stat5b
59-CCTAACTTGCTCTTGGACAGG-39
59-CAGCAGCAGGTCATAGAGAGG-39
59-GCCAATCATGTGCACCAGTTCCTT-39
59-AAAGCCAAGAGAAACGGTGGGCAT-39
59-CCCACAGGAGTTTGTCAAGCTC-39
59-TAACTTCAGACATCATTTCCGG-39
59-CCCATTAGAAAACACTGACCG-39
59-GGGAACACGAATGAGGATTGT-39
59-AAAGTATCTTGTCCAGACTCGCTG-39
59-AGCAGTTCTTCAGACTTGCCCTT-39
59-AGGGGACTTAATGAGGTCGTA-39
59-TAGCTCCAGCGAGATTAGAGT-39
59-TCACAGAGCAGAGAATGGACT-39
59-GTATGGCTGATACCACAAGGT-39
59-TTCGAGCAGAACTCATGGCTA-39
59-CAACCAGGTAGAATGCCATCT-39
59-GGATAAACCTGTACCACTTCC-39
59-CTTTGATGAAGTCCTGAGCTAC-39
59-GTCTTCAGACCAGAAACCAAG-39
59-CCTTAAGGCACAAGTATGCAG-39
59-GATTCTTGTGGATGGGGATTG-39
59-GTCCAGCATGATGTGTCTCAT-39
59-AAGCAGGCGACGGGAACAAGA-39
59-AAGACATGATTGGGTGGATAC-39
59-CAGAACGAGGTTGTAAGCCAT-39
59-ATCTTCAGCACAAAGCCATCT-39
References
344
7
203
8
325
9
462
10
279
11
425
12
480
13
440
14
366
15
447
16
401
17
406
18
272
19
For mRNA analysis by RT-PCR, oligonucleotide primers were designed based on the cDNA sequence. PRL-RL, PRL receptor-long form; LH-R,
LH receptor; IL-6R, IL-6 receptor.
sensitive luteal cell line in the study of luteal cell differentiation and gene regulation.
Materials and Methods
Materials
McCoy’s 5A-Ham’s F12 (1:1) medium, d-glucose, 17-b estradiol,
8-bromo-cAMP, and cycloheximide were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI-1640 medium, antibiotic-antimycotic solution, nonessential amino acids, and sodium pyruvate were from Mediatech
(Washington, D.C.). FBS was from HyClone (Logan, UT). [a-32P]deoxycytidine triphosphate (dCTP) was from Amersham (Arlington Heights, IL).
Twenty-five- or 75-cm2 culture flasks were from Becton Dickinson Co.
(Franklin Lakes, NJ). Taq DNA polymerase was from Perkin-Elmer Co.
(Foster City, CA). GeneScreen nylon membranes were from New England
Nuclear Systems (Boston, MA). Lipofectin and G418 sulfate (Geneticin)
were from Life Technologies Inc. (Grand Island, NY).
Animals
Pregnant Sprague-Dawley rats (day 1 5 sperm positive) purchased
from Sasco Animal Laboratories (Madison, WI) were housed at 24 C with
a 14-h light, 10-h dark cycle and allowed free access to Purina rat chow
and water. The care and handling of the rats conformed with the NIH
guidelines for animal research. The experimental protocol was approved
by the Institutional Animal Care and Use Committee.
Transformation of rat luteal cells by SV-40
Large luteal cells were purified to homogeneity by flow cytometry
from the corpus luteum of day 14 pregnant rats as reported previously
(3). Cells were cultured in medium (McCoy’s 5A-Ham’s F12, 1:1) containing 25 mm HEPES, 2% antibiotic-antimycotic solution, and 5% FCS.
After plating for 24 h, the cells were washed several times and then
infected with the SV-40 tsA209 mutant virus as previously reported (4).
Transformed cells were maintained at the permissive temperature (33 C)
until colonies were identified. Several colonies of the transformed cells
were isolated and passaged. One clone, designated GG-CL cells, was
FIG. 1. Growth curve of GG-CL cells. A group of cells (105) were
plated in a 25-cm2 tissue culture flask and cultured at 33 C for 10 days.
Another group of cells was cultured at 33 C for 2 days and was then
shifted to 40 C and cultured for 10 days. Cells were harvested at each
time point by trypsinization and counted.
used in this study. The GG-CL cells were cultured in a 25- or 75-cm2 flask
with the incubation medium (RPMI-1640 medium containing 23 antibiotic-antimycotic solution, 13 nonessential amino acids, 13 sodium
pyruvate, 0.5% d-glucose, and 10% FBS) at the permissive (33 C) and the
nonpermissive (40 C) temperatures under an atmosphere consisting of
5% CO2-95% air.
Transfection of GG-CL cells with the PRL receptor
For the stable transfection of GG-CL cells with the PRL receptor, we
adopted the procedure of Felgner et al. (5) with slight modifications.
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RAT LUTEAL CELL LINE
GG-CL cells were plated in six-well Falcon dishes and grown at 33 C to
33% confluency in RPMI-1640 medium containing 5% FBS. Cells were
washed with serum-free medium with no antibiotics and were transfected using Lipofectin according to the manufacturer’s protocol. The
Endo • 1998
Vol 139 • No 4
cells were transfected with 10 mg of the expression vector pMT2poly
containing the PRL receptor complementary DNA (cDNA) and with
pSV2neo vector, both generously provided by Dr. Daniel Linzer (Northwestern University, Chicago, IL). After transfection the medium was
replaced with the growth medium containing 5% FBS and antibiotics
and incubated for 48 h. After 48 h, medium was again replaced with fresh
growth medium and treated with 100 mg/ml of G418 sulfate. G418
sulfate addition was continued every alternate day until G418 sulfateresistant colonies were identified. These colonies were picked and cultured in the growth medium containing 5% FBS until the cells were
confluent. For identifying the successful stable transfection with PRL
receptor, cells were grown and passaged several times. PRL receptor
messenger RNA (mRNA) expression in these cells was determined by
RT-PCR using PRL receptor-specific primers.
Treatment of GG-CL cells
To examine the effects of estradiol on ER-b mRNA expression and of
cAMP on the orphan nuclear receptor nur 77 mRNA expression, GG-CL
cells were cultured at 33 C until 50% confluent and shifted to 40 C for
2 days. Cells were then treated with either 17b-estradiol for 6 h, or with
8-bromo-cAMP (100 mm) in the presence or absence of cycloheximide (10
mg/ml) for either 30, 60, or 120 min. To examine the effect of PRL on
20a-HSD mRNA expression in GG-CL cells, GG-CL cells transfected
with PRL receptor were cultured at 33 C until 50% confluent and then
shifted to 40 C for 2 days. Cells were then treated with ovine PRL (1
mg/ml; NIDDK oPRL-20, 31 IU/mg) for either 4, 8, 24, or 48 h. After
culture, the cells were washed with PBS several times and stored at 280
C for RNA isolation.
Isolation of total RNA and RT-PCR
FIG. 2. Morphology of GG-CL cells. Cells cultured at 33 C near confluence seemed to be overlapped and binucleated (upper panel). Cells
cultured at 40 C seemed to be monolayered, differentiated, and mononucleated (lower panel). Magnification, 6003.
Total RNA from the cells was isolated by the guanidinium-isothiocyanate-phenol-chloroform extraction procedure (6). For mRNA analysis by RT-PCR, oligonucleotide primers shown in Table 1 were designed based on each cDNA sequence. Each reaction also included
primers (59-CTGAAGGTCAAAGGGAATGTG-39 and 59-GGACAGAGTCTTGATGATCTC-39) to amplify ribosomal protein L19 or primers (59-CGTTCACCTTGATGAGCCCATT-39 and 59-TCCAAGGGTCCGCTGCAGTC-39) to amplify ribosomal protein S16. Both L19 and S16
were used as internal controls (20, 21). The predicted size of the PCRamplified product was 194 bp for L19 and 100 bp for S16. One to 3 mg
of total RNA were reverse-transcribed at 42 C in a 20-ml reaction mixture
[1 3 PCR buffer, 2.5 mm deoxynucleoside triphosphates, 5 mm random
hexamer primers, 1.5 mm MgCl2, and 200 U Moloney murine leukemia
virus reverse transcriptase (Life Technologies)]. For PCR amplification,
a mixture containing oligonucleotide primers (sequences shown in Table
1; 50 pmol), [a-32P]dCTP (2 mCi at 3000 Ci/mmol), and Taq DNA polymerase (2.5 U) was added to each reaction. The total volume was in-
FIG. 3. Expression of ER-b mRNA (A) and effects of estradiol on ER-b mRNA expression in GG-CL cells (B and C). Total RNA was isolated
from GG-CL cells either cultured at 33 C and 40 C (A) or treated with 17b-estradiol for 6 h at 40 C (B and C) and then subjected to RT-PCR
as described in Materials and Methods. The quantification data are expressed as a percentage of control and mean 6 SEM of three different
experiments.
RAT LUTEAL CELL LINE
creased to 90 ml with 1 3 PCR buffer, and the samples were overlaid with
mineral oil. Amplification was carried out for 30 cycles using a 65 C
annealing temperature in a Perkin-Elmer/Cetus (Norwalk, CT) thermal
cycler. The conditions were such that amplification of the product was
in the exponential phase, and the assay was linear with respect to the
amount of input RNA. Reaction products were electrophoresed on a 8%
polyacrylamide nondenaturing gel. Each RT-PCR reaction included L19
or S16 ribosomal protein mRNA primers to normalize the data. After
autoradiography, data were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Northern blot analysis
Equal amounts (20 mg) of RNA as determined by absorbance at 260
nm were loaded, and the equivalency was verified by ethidium bromidestained 18S and 28S ribosomal RNA bands. RNA was fractionated
through a 1% agarose gel containing 0.74 m formaldehyde and was
transferred to a GeneScreen nylon membrane by overnight capillary
blotting with 103 sodium chloride-sodium citrate (SCC buffer; 13 5 150
mm sodium chloride and 15 mm sodium citrate, pH 7.0). Membranes
were baked at 80 C under a vacuum for 2 h to immobilize the RNA on
the membranes. A rat nur 77 cDNA probe (kindly provided by Dr. Lester
F. Lau, the University of Illinois at Chicago, Chicago, IL) was radiolabeled by random priming method (22) using [a-32P]dCTP (3000 Ci/
mmol) and was purified from the unincorporated radionucleotide by
column chromatography. The blots were prehybridized and hybridized
in a buffer containing 50% formamide, 0.6 m NaCl, 1 mm EDTA, 25 mm
HEPES (pH 6.5), 50 mm sodium phosphate (pH 6.5), 0.1% SDS, 53
Denhardt’s solution, 10% dextran sulfate (hybridization only), and
salmon sperm DNA (100 mg/ml) at 42 C for 18 h. Blots were washed with
23 SSC (containing 0.1% SDS) for 10 min, 13 SSC (containing 0.1% SDS)
FIG. 4. Expression of PR (A) and GR (B) mRNA in GG-CL cells. Total
RNA was isolated from GG-CL cells cultured at 33 C and 40 C and
subjected to RT-PCR as described in Materials and Methods. Decidua
(DT) of day 10 pseudopregnant rats was used as a positive control for
PR. The data are representative of more than three different experiments.
1939
for 10 min, and 0.53 SSC (containing 0.1% SDS) for 5 min at 68 C. The
resultant blots were exposed to Kodak X-Omat film (Eastman Kodak
Co., Rochester, NY) using intensifying screens at 280 C.
Results
We observed several colonies 6 months after transfection
with a temperature-sensitive mutant of SV-40 (tsA 209). One
of the clones (GG-CL) was chosen and grown at 33 C for
further characterization. Propagation to several passages did
not affect the cellular growth rate at 33 C, thus confirming the
stability of this cell line.
To demonstrate that GG-CL cells are indeed temperature
sensitive, cells were plated at the density of 105 cells per flask
in a 25-cm2 tissue culture flask and cultured at 33 C for 10
days. Cells were also cultured at 33 C for 2 days and were
then shifted to 40 C and cultured for 10 days. Cells were
harvested at each time point by trypsinization and counted
by the trypan blue dye exclusion method. At 33 C, cells
rapidly divided and became confluent, whereas cells that
were moved to 40 C ceased to divide and were able to grow
only as monolayers (Fig. 1).
As shown in Fig. 2, GG-CL cells exhibited remarkable
morphological change when they were shifted from 33 C to
40 C. At 33 C, the cells seemed to be small and compact and
showed a lower cytoplasm-to-nucleus ratio (Fig. 2, upper
panel). At 40 C, these cells assumed a monolayer appearance;
they became differentiated and flat, with an increased cytoplasm-to-nucleus ratio and a typical appearance resembling
the primary culture (Fig. 2, lower panel).
To determine whether GG-CL cells express the genes
found in normal luteal cells, mRNA expression was examined by either Northern analysis or RT-PCR with primers
specific to each mRNA. Since the corpus luteum is highly
responsive to estradiol, we first examined whether these cells
express ERa or the newly identified ER-b (8). ERa mRNA
could not be detected in these cells by RT-PCR (data not
shown), whereas ER-b mRNA was clearly expressed (Fig.
3A). The level of expression of this receptor was increased at
40 C when the cells became fully differentiated (Fig. 3A). Since
estrogen has been reported to modulate ER levels in several
estrogen target tissues, such as the uterus and mammary gland
(23–25), we examined whether estradiol can influence the ex-
FIG. 5. Expression of interleukin-6 receptor (IL-6R) (A), PRL receptor-long form (PRL-RL) (B) and LH receptor (LH-R) (C) mRNA in GG-CL
cells. Total RNA was isolated from GG-CL cells cultured at 33 C and 40 C and subjected to RT-PCR as described in Materials and Methods.
Corpora lutea (CL) of day 15 pregnant rats were used as positive controls for LH-R and PRL-RL. The data are representative of more than three
different experiments.
1940
RAT LUTEAL CELL LINE
pression of ER-b in this luteal cell-derived cell line. As shown
in Fig. 3, B and C, the GG-CL cells responded to the estradiol
challenge with a dose-dependent increase in ER-b mRNA expression. The rat corpus luteum does not express the progesterone receptor (PR) but does express the GR (2, 26), and we
recently have shown that the tropic action of progesterone in the
corpus luteum is through the GR (2). Indeed, as reported previously (2), no PR could be detected in the GG-CL cells (Fig. 4A)
whereas the GR mRNA was expressed at 33 C and was markedly increased at 40 C (Fig. 4B). Once we established the expression of steroid receptors in these cells, we examined for
membrane receptors known to be highly expressed in the rat
corpus luteum: the PRL receptor and LH receptor. We also
examined for IL-6 receptor expression since this receptor was
recently shown to be expressed and regulated in the rat corpus
luteum (27). As shown in Fig. 5A, IL-6 receptor mRNA was
FIG. 6. Effect of cAMP on nur 77 mRNA expression in GG-CL cells.
GG-CL cells were cultured with 8-bromo-cAMP (100 mM) at 40 C for
either 30, 60, or 120 min in the presence or absence of cycloheximide
(CHX; 10 mg/ml). Total RNA was isolated on each time of culture and
subjected to Northern blot analysis as described in Materials and
Methods. The data are representative of three different experiments.
FIG. 7. Expression of 20a-hydroxysteroid dehydrogenase (20a-HSD) (A),
cholesterol side chain cleavage cytochrome P450 (P450SCC) (B), 3b-hydroxysteroid dehydrogenase (3b-HSD)
(C), and aromatase cytochrome P450
(P450arom) (D) mRNAs in GG-CL cells.
Total RNA was isolated from GG-CL
cells cultured at 33 C and 40 C and
subjected to RT-PCR as described in
Materials and Methods. Corpora lutea
(CL) of day 15 pregnant rats were used
as positive controls for P450SCC, 3bHSD, and P450arom. The data are representative of more than three different
experiments.
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Vol 139 • No 4
readily expressed in the GG-CL cells at both temperatures.
However, neither the PRL receptor (Fig. 5B) nor the LH receptor
mRNA (Fig. 5C) could be detected in these cells although they
were highly expressed in the corpus luteum used as a control.
Although the GG-CL cells did not express the LH receptor, they
did respond to cAMP stimulation with an increase in nur 77
mRNA expression. Nur 77, also known as NGFI-b, is an immediate-early gene whose expression is stimulated by cAMP
and whose mRNA is stabilized by cycloheximide (28, 29). As
shown in Fig. 6, cAMP caused a rapid induction of nur 77
mRNA. No nur 77 expression could be detected in GG-CL cells
untreated with cAMP (lane 1). However, within 30 min of
cAMP treatment, nur 77 mRNA became expressed (lane 2) and
reached peak value at 60 min (lane 4). A marked decline was
seen thereafter (lane 6). Treatment with cycloheximide caused
a superinduction of nur 77 mRNA (lanes 3, 5, and 7).
To determine whether the steroidogenic capacity of the
corpus luteum is retained in GG-CL cells, we examined the
expression of genes for the steroidogenic enzymes P450scc,
3b-HSD, and P450 aromatase. In addition, because the corpus luteum expresses 20a-HSD, a gene that we recently
cloned (14) and showed to be under the control of PRL (30,
31), we have also examined the expression of this enzyme in
GG-CL cells maintained at both temperatures. As shown in
Fig. 7, B, C, and D, P450scc, 3b-HSD, and P450 aromatase
mRNAs were undetectable in GG-CL cells, although they
were highly expressed in the corpus luteum used as a control.
However, 20a-HSD mRNA was expressed at both temperatures with much higher expression when the cells were
differentiated at 40 C (Fig. 7A).
Since GG-CL cells express 20a-HSD, we examined
whether these cells express the JAK2/Stat5b system known
to mediate PRL signaling in many cells including luteal cells
RAT LUTEAL CELL LINE
FIG. 8. Expression of JAK2 and Stat5b mRNAs in GG-CL cells. Total
RNA was isolated from GG-CL cells cultured at 40 C and subjected
to RT-PCR as described in Materials and Methods. Rat liver, well
known to be rich in JAK2 and Stat5b, was used as a positive control.
The data are representative of three different experiments.
FIG. 9. Effect of PRL on 20a-HSD mRNA expression in GG-CL cells
transfected with the PRL receptor. GG-CL cells were transfected with
the PRL receptor (PRL-R) (A), and they were cultured with PRL (1
mg/ml) at 40 C for either 4, 8, 24, or 48 h (B). Total RNA was isolated
on each time of culture and subjected to RT-PCR as described in
Materials and Methods. The data are representative of three different
experiments.
(31–35). We used liver tissue as a control, which is known to
highly express the JAK2 kinase and the Stat5 transcriptional
factors (36). As shown in Fig. 8, the GG-CL cells readily
expressed both JAK2 and Stat5b mRNA. We therefore transfected these cells with the PRL receptor and examined
whether they become responsive to PRL. The results shown
in Fig. 9 indicate that the cells transfected with the PRL
receptor (Fig. 9A) respond to PRL with an inhibition in 20aHSD mRNA expression (Fig. 9B).
Discussion
This is, to our knowledge, the first successful development
of a cell line originating from the large luteal cells of the rat
corpus luteum that retains many of the cell-specific elements
encountered in the primary cells of origin. Luteal cells are
terminally differentiated and fail to proliferate in primary
culture. Their transfection with a temperature-sensitive mutant (tsA209) of the SV-40 virus endows these cells with the
ability to grow in an unrestricted manner. The tsA mutant of
SV-40 are mutant viruses, which are defective in the A gene
1941
required for the maintenance of the transformed phenotype
in the mammalian cells (4, 37). Therefore, the tsA mutantinfected luteal cells are conditionally transformed cells and
express the transformed phenotype only at the permissive
temperature (33 C). At the nonpermissive temperature (40
C), these cells revert to the normal morphological differentiated phenotype similar to that of primary luteal cells in
culture. The ability of the luteal cell line to divide at 33 C and
to differentiate at 40 C allows the use of these cells without
the complication of increases in cell number.
The GG-CL cells express many genes characteristic to the
corpus luteum, although they have lost their ability to produce progesterone upon transformation. However, this lack
of progesterone production has proven to be an asset in our
attempts to determine the effect of progesterone on the corpus luteum. For years we were trying to determine whether
progesterone can act as a local luteotropin. The absence of PR
in the rat corpus luteum, as well as the copious amounts of
progesterone produced by the primary luteal cells themselves, has hampered these investigations. Very recently,
using this cell line, we were able to demonstrate that progesterone acts through the GR and decreases the expression
of 20a-HSD, an enzyme responsible for the catabolism of
progesterone (2).
Estradiol plays an important role in the regulation of the
luteal function (38). Our results indicate that the GG-CL cells
express the ER-b but not ER-a. The ER-b was recently shown
to be expressed predominantly in the ovary (8, 39) and corpus luteum (40), and this is, to the best of our knowledge, the
first cell line described that expresses only ER-b. The ER-b in
the GG-CL cell appears to be functional and to mediate
estradiol action since estradiol treatment caused a dose-dependent increase in ER-b mRNA levels. It seems therefore
that GG-CL cells confer a unique model by which to study
and elucidate ER-b regulation and function independently
from the ER-a.
Another focus of our laboratory is the regulation of 20aHSD by PRL (14, 30, 31). The GG-CL cells express 20a-HSD
mRNA endogenously, as well as the tyrosine kinase JAK2
and the transcriptional factor Stat5b. The latter two molecules are known to be involved in PRL signaling in many
cells, including luteal cells (31–35). However, no PRL receptor mRNA could be detected in GG-CL cells. We have therefore transfected the GG-CL cells with the PRL receptor. The
cells transfected with the PRL receptor responded to PRL
treatment with an inhibition of 20a-HSD mRNA expression,
similar to the cells of origin.
Although the GG-CL cells do not express the LH receptor,
they do respond to cAMP with a rapid increase in the expression of the immediate-early gene nur 77 (28, 29). In
addition, the superinduction of nur 77 mRNA caused by
cycloheximide indicates the presence of proteins involved in
nur 77 mRNA degradation in GG-CL cells. We have chosen
to examine the effect of cAMP on nur 77 in these cells because
we have recently demonstrated two response elements for
this transcription factor in the 20a-HSD promoter (41). This
orphan nuclear receptor is a potent transcriptional activator
of the P450 c21 (28) and P450 c17 genes (42). The finding that
cAMP up-regulates 20a-HSD in ovarian cells (43, 44), together with the cAMP mediated up-regulation of nur 77 and
1942
RAT LUTEAL CELL LINE
the response elements present in the 20a-HSD promoter,
raises the interesting possibility that nur 77 may stimulate
20a-HSD gene expression.
In summary, we have successfully established a cell line
derived from the large luteal cells of the rat corpus luteum.
These cells showed a temperature-dependent phenotype
with respect to morphology and growth. They also express
key genes encoding enzymes and receptors inherent to this
defined luteal cell population and respond to stimulation by
estradiol, PRL, and cAMP.
18.
19.
20.
21.
22.
Acknowledgments
23.
We are grateful to Dr. Daniel Linzer for the PRL receptor expression
vector, to Dr. Lester F. Lau for the nur 77 cDNA, and to The National
Institute of Diabetes and Digestive and Kidney Diseases and National
Hormone and Pituitary Program (NIH) for the ovine PRL. We also thank
Linda Alaniz for her photographic work, Rosemary Clepper for animal
care, and Vivian Rogala for assistance in the manuscript preparation.
24.
References
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