ORIGINAL ARTICLE
Establishment of an immortalized porcine granulosa cell line
(PGV) and the study on the potential mechanisms of
PGV cell proliferation
Ming-Te Lin
Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on November 18, 2004)(Revised for publication on December 15, 2004)(Accepted for publication on December 16, 2004)

Abstract. In order to establish an immortalized granulosa cell line and to investigate the potential
mechanisms of immortalized cell proliferation, simian virus (SV) 40 was used to infect porcine granulosa cells from small follicles (1–2 mm in diameter), and one colony was selected after four weeks ofculture. The colony was digested with trypsin and the cells were cultured for more than 300 days
(named PGV). The SV40 large T antigen gene and its products were conﬁrmed in immortalized cells
by Southern blotting and immunohistochemistry. Progesterone production was not detected in theconditioned culture media with follicle-stimulating hormone (FSH) and forskolin, possibly due to thelack of P450scc gene transcription as examined by Northern blotting. PGV cells responded signiﬁcantly
to the stimulation of sera (fetal bovine and horse sera) and protein kinase C (PKC) stimulators (PMAand OAG), while PKC inhibitors (staurosporine and calphostin C) blocked both sera and PKC stimulation. Phospholipase C (PLC) and phosphatidic acid phosphatase (PAP) inhibitors (U73122 andpropranolol) signiﬁcantly reduced PGV cell proliferation, while PMA restored PLC and PAP inhibition.
These data suggest that diacylglycerol (DAG) is produced in PGV cells by PLD as well as by PLC, andthat DAG then activates PKC stimulating the PGV cell cycle through yet unknown mechanisms. Thus,an immortalized granulosa cell line is very useful to study granulosa cells in vitro, as the cells arehomogeneous and are a functionally deﬁned population. (Keio J Med 54 (1): 29–38, March 2005)
Key words: granulosa, immortalization, PKC, PLC, PLD

granulosa cell proliferation are still poorly understood.
Since granulosa cells luteinize spontaneously in vitro
and do not undergo more than one cell doubling in
culture,2 it is difﬁcult to study the mechanisms of granulosa cell proliferation using cell cultures. Moreover,in order to study granulosa cells in vitro, ovaries must
be collected from animals whenever a study is set
up. In addition, granulosa cell populations obtained
from ovaries are heterogeneous and less than 50% of
the cells are viable. Thus, it is difﬁcult to obtain pure,functionally-deﬁned populations. Hence, it is advantageous to develop a homogeneous population of granulosa cells.
In this study an immortalized porcine granulosa cellline (named PGV) was established with simian virus(SV) 40 infection, and was used to investigate the potential mechanisms underlying the regulation of gran-Introduction
During ovarian follicle maturation from a primordial
follicle to a preovulatory follicle, various events occur
both morphologically and functionally inside the follicle. Among these events, the increase in the number ofthe granulosa cells plays a central role in follicle development.1 If a developing human follicle is selected
to ovulate it may grow 50-fold, increasing from 0.4 mm
to 20 mm in diameter; the growth of the Grafﬁan follicleis accompanied by granulosa cell proliferation andfollicular ﬂuid accumulation in the antrum. It is also
well known that gonadotropins, especially folliclestimulating hormone (FSH), govern follicle growth invivo. Although studies on steroidogenesis in the follicles
in vivo or in cultured granulosa cells in vitro have been
performed extensively to date, the exact mechanisms of

Reprint requests to: Dr. Ming-Te Lin, Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi,
Shinjukuku, Tokyo 160-8582, Japan
29

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Lin M-T: Immortalized granulosa cell proliferation

ulosa cell proliferation. PGV cell numbers increased
with sera and also with protein kinase C (PKC) stimulators, but decreased with PKC inhibitors. Neither
PKA stimulators nor growth factors increased PGV cellnumbers. Both phospholipase C (PLC) and phosphatidic acid phosphatase (PAP) inhibitors reduced PGVcell proliferation, while PKC stimulator restored PLC
and PAP inhibition. Based upon these data, it may beconcluded that the PKC pathway is stimulated by diacylglycerol (DAG) generated through the PLD as wellas PLC pathways and that the PKC pathway may play
an important role in PGV cell proliferation as well as inproliferation of primary granulosa cells.

a plasmid containing the SV40 whole genome wasdigested with restriction enzymes (Stu I and BamH I)
and the fragment (2657 bp) corresponding to large T
antigen was puriﬁed and radiolabeled with 32 P. Total
genomic DNA was extracted from PGV cells anddigested with HindIII. Southern blotting was carriedout with the radiolabeled probe after agarose-gel electrophoresis.
To conﬁrm the existence of large T antigen products
immunohistochemically, PGV cells were grown on
cover glasses for two days, ﬁxed with ethanol andstained with large T antigen-speciﬁc primary antibody(Wako, Japan) followed by FITC-labeled secondary
antibody against mouse IgG (Wako, Japan).

Materials and Methods
Porcine granulosa cell cultures
Porcine ovaries were collected at a local slaughterhouse in saline with penicillin (50 mg/ml) and streptomycin (50 mg/ml, Gibco, USA). Porcine granulosa cells
were aspirated from small follicles (1–2 mm in diameter) with a 23-gauge needle and syringe, centrifuged
three times, and then re-suspended with TCM199 containing 10% fetal bovine serum (FBS, Gibco, USA),
penicillin (50 mg/ml), streptomycin (50 mg/ml), fungizone (0.25 mg/ml, Gibco) and gentamycin (50 mg/ml,
Essex Japan, Japan) according to the methods by
Channing et al.3,4 Aliquots of cells (5  10 5 viable cells/well) were plated to 35 mm dishes and incubated at 5%
CO2 in air.
Infection of SV40 and establishment of a cell line
After two days of initial culture, SV40 was used to
infect the cultured porcine granulosa cells at 10 4 plaqueforming units (pfu)/ml, 10 5 pfu/ml and 10 6 pfu/ml; twodishes of mock infection were used as controls. Twohours following infection, cells were vigorously washedseveral times with fresh medium; thereafter, media
were changed every two days. Four weeks after infection one colony was selected in a dish with 10 6 pfu/ml
of SV40. The colony was digested with trypsin andthe cells were transferred to another dish. When cells
reached sub-conﬂuence, cells were treated with trypsin
and cell numbers were counted with a hemocytometer.
Population doublings (PDS) were calculated at every
cell passage. The cell line was named PGV (porcine
granulosa cell-virus transformed).
Conﬁrmation of the existence of SV40 large T antigen in
PGV cells
The SV40 genome encoding the large T antigen
was conﬁrmed by Southern blotting. To create a probe,

Measurement of progesterone concentrations in
conditioned culture media and cyclic adenosine 3 0 ,5 0 monophosphate (cAMP) in PGV cells and primary
granulosa cells
PGV cells (1  10 6 cells/well) and primary granulosa
cells (1  10 6 viable cells/well) were inoculated to 35
mm dishes and incubated for two days with 100 ng/ml
FSH (AFP2844B, provided by National Hormone andPituitary Program, NIH, USA) and forskolin (0.5 mM)
with IBMX (3-isobutyl-1-methylxanthine, 105 M).
Progesterone concentrations in conditioned media andcAMP in the cells were determined by their speciﬁcradio-immunoassays (RIA) (DPC Japan, Japan, andAmersham, UK, respectively). The inter-assay andintra-assay coefﬁcients of variations in progesterone
and cAMP were 8.5% and 6.2%, and 8.7% and 6.8%,
respectively. All chemicals used in this study werepurchased from Sigma, USA except where otherwise
noted.
Determination of the P450scc mRNA by Northern blot
analysis
In order to investigate the expression of the P450scc(cytochrome P450 side-chain cleavage enzyme) genein PGV cells, Northern blotting was carried out using
bovine P450scc cDNA (provided by Prof. Yoshiaki
Fujii, Tohoku University) after extraction of total RNA
from PGV cells and agarose-gel electrophoresis using astandard protocol.
Reversibility of steroidogenic activity in PGV cells withretinoic acid
As PGV cells possess no steroidogenic activity, PGV
cells were cultured with retinoic acid (1013 M@105 M)
for up to 2 weeks and then challenged with FSH (30 ng/ml), luteinizing hormone (LH) (AFP4345B, provided
by NIH, 10 ng/ml) and forskolin (0.5 mM) (þIBMX,

Keio J Med 2005; 54 (1): 29–38

31

105 M). Progesterone concentrations in conditioned
media were measured by RIA after 2 days of culture.
PGV cell proliferation investigations
In order to investigate the effects of intracellularsignal transduction mechanisms of PGV cells on cellproliferation, various concentrations of sera (FBS andhorse serum), gonadotropins (FSH and LH), growth
factors (insulin-like growth factor (IGF)-I and -II (provided by Fujisawa Pharmaceutical Co., Japan), insulin
and epidermal growth factor (EGF), tyrosine kinase
inhibitor (genistein), PKA stimulators (forskolin, 8bromo-cAMP, and dibutyryl cAMP), PKC stimulators (phorbol-12-myristate-13-acetate (PMA) and1-oleoyl-2-acetyl-glycerol (OAG), PKC inhibitors(staurosporine and calphostin C), calcium ionophore(A23187), calmodulin antagonists (W5 and W7), PLC
inhibitor (U73122), and PAP inhibitor (propranolol)
were added to PGV cells (1  10 5 cells/well) in 24
multi-well dishes in replicate conditions. After threedays of culture, except where otherwise stated, cellnumbers were counted with hemocytometer using
0.066% trypan blue. As maximum doses of respective
vehicles in the cultures did not inﬂuence PGV cellnumbers, only medium containing 10% horse serum
was used as a control.
Each experiment was performed at least threetimes and representative data were shown. Results are
expressed as the mean G standard error of mean (SE).
Statistical analysis
One-way analysis of variance (ANOVA) followed by
Duncan’s new multiple range tests was used to test for
statistical signiﬁcance ( p < 0:05) between treatments.5
Different superscripts in both table and ﬁgures denotethe signiﬁcant difference ( p < 0:05) between the treatments.

Fig. 1 Morphology of immortalized porcine granulosa cells (PGV)
infected with SV40. Cells were elongated with ﬁbroblast-like cell
contours with relatively large nuclei.

still viable on the 27th day of culture, many vacuoles
appeared within the cells by the 43rd day, and almost
all primary cells were not viable by the 45th day (datanot shown) in contrast to the fact that all PGV cells
were viable even on the 300th day.
Conﬁrmation of large T antigen in PGV cells
Southern blotting was performed to conﬁrm thepresence of SV40 large T antigen DNA in PGV cells.
The hybridized DNA fragments (1768 bp, 1169 bp, and526 bp) with HindIII digestion were compatible inlength to those predicted with HindIII digestion of
SV40.
SV40 large T antigen products could also be visualized immunohistochemically after treatment with antilarge T antigen antibody, followed by FITC-labeledsecond antibody (data not shown).

Results
Establishment of an immortalized cell line (PGV)

Measurement of progesterone concentrations in conditioned culture media and cAMP in cells

Porcine granulosa cells from small follicles were
infected with SV40. After four weeks of culture one
colony was selected in a dish with 10 6 pfu/well of SV40.
The colony was digested with trypsin, and maintainedin culture. When cell numbers and PDS were calculated
at each passage up to 300 days, cell numbers increased
about sixteen times during one week. PGV cells exhibited an elongated and ﬁbroblastic shape with relatively
large nuclei as shown in Fig. 1.
Primary porcine granulosa cells and PGV cells weremaintained in culture; although the primary cells were

Progesterone concentrations in conditioned culture
media and cAMP contents in cells were determinedby their speciﬁc RIAs. Although progesterone concentrations when challenged with FSH and forskolin weresigniﬁcantly higher than control levels in primary granulosa cell cultures, progesterone concentrations in PGVcell cultures were not detectable even by addition of
FSH and forskolin as shown in Table 1. cAMP contents
in both primary cells and PGV cells were slightly higher
when treated with 100 ng/ml FSH than controls, but thedifference was not signiﬁcant. However, cAMP contents

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Lin M-T: Immortalized granulosa cell proliferation

when cultured with forskolin (þIBMX) were significantly higher than controls in both cell types.

Northern blotting was carried out utilizing P450scc
cDNA. However, P450scc mRNA could not be detectedin PGV cells (data not shown).

Determination of P450scc mRNA in PGV cells
To conﬁrm the expression of the P450scc (ratelimiting enzyme of steroidogenesis) gene in PGV cells,
Table 1 Effect of FSH and forskolin on progesterone and cyclic
AMP synthesis by primary granulosa cells and PGV cells. After 2 daysof culture with FSH (100 ng/ml) and forskolin (0.5 mM) (þIBMX,
105 M), progesterone concentrations in conditioned culture media
and cAMP contents in cells were determined by their speciﬁc RIAs.
Different superscripts show signiﬁcant differences ( p < 0:05 by
ANOVA and Duncan’s new multiple range test).
Addition

Cell typePrimary

To investigate whether PGV cells could restore
steroidogenic activity, retinoic acid (1013 M@105 M)
was added to PGV cells for up to 2 weeks. The cells
were then incubated with FSH (30 ng/ml), LH (10 ng/ml) and forskolin (0.5 mM) (þIBMX, 105 M), andprogesterone concentrations were measured in conditioned culture media by RIA. No progesterone could
be detected in all culture dishes (data not shown).
PGV cell proliferation investigations

PGV
6

None
FSH (100 ng/ml)
Forskolin (0.5 mM)

hProgesterone synthesisi
212 G 17a
2073 G 166b
4654 G 372c

n mol/ml  10 cells
ND
ND
ND

None
FSH (100 ng/ml)
Forskolin (0.5 mM)

hcAMP formationi
1.2 G 0.2a
1.7 G 0.3a
7.2 G 0.7b

p mol/ml  10 6 cells
1.4 G 0.3a
1.7 G 0.3a
11.8 G 0.8b

mean G SE, ND: Not Detectable.

Reversibility of steroidogenic activity with retinoic acid

Serum: In order to see whether FBS and horse serum(HS) stimulated PGV cell numbers, FBS (1%@20%)
and HS (10%) were added to PGV cell cultures. Cell
numbers were counted with hemocytometer every twodays up until six days.
Although PGV cell numbers in serum-free medium(control) increased on day 4, they decreased on day
6 as shown in Fig. 2. FBS signiﬁcantly stimulated PGVcell numbers in a dose- and time-dependent manner.
Cell numbers in 1% FBS were not signiﬁcantly different

Fig. 2 Effects of increasing concentrations of fetal bovine serum (FBS) and horse serum (HS) on PGV cell number (/well). PGV cells (1 
10 5 cells/well) were cultured in 24 multi-well dishes with FBS (1%@20%) and HS (10%) for 2 to 6 days, and cell numbers were counted withhemocytometer using 0.066% trypan blue as described in Materials and Methods. Each column and bar represent the mean G SE, and different
superscripts (a@f) show signiﬁcant differences between groups ( p < 0:05 by ANOVA and Duncan’s new multiple range test). Panel A, on day 2
PGV cell numbers with greater than 5% FBS were signiﬁcantly higher than controls (serum-free media). Cell numbers with 10% HS were equal
to those with 15% and 20% FBS. Panel B, on day 4 PGV cell numbers with 1% FBS were signiﬁcantly higher than controls. Cell numbers withall concentrations of FBS were higher on day 4 than on day 2. Cell numbers with 10% HS were signiﬁcantly less than those at 1% FBS. Panel C,
on day 6 FBS further stimulated PGV cell proliferation. There was no signiﬁcant difference in cell numbers between 15% and 20% FBS even on
day 6. Control cell numbers were less than those on day 4.

Keio J Med 2005; 54 (1): 29–38

33

from controls on day two (Fig. 2-A), while they weresigniﬁcantly higher on days 4 and 6 (Fig. 2-B and 2-C).
There was no signiﬁcant difference in cell numbers between 15 and 20% FBS on each day. PGV cell numbers
in 10% HS were nearly equal to those with both 15 and
20% FBS on day 2 (Fig. 2-A), while on days 4 and 6
they were signiﬁcantly less than those with 1% FBS(Fig. 2-B and 2-C).
Gonadotropins: FSH (30 ng/ml) and LH (10 ng/ml)
were added to PGV cell cultures and cell numbers werecounted with hemocytometer after 3 days. There wasno signiﬁcant difference between controls and treatedcells (data not shown).
Growth factors and tyrosine kinase inhibitor: EGF(0, 1, 10, 100 ng/ml), IGF-I (0, 1, 10, 100 ng/ml) and -II(0, 1, 10, 100 ng/ml), insulin (0, 0.5, 1, 2, 5, 10 ng/ml),
and the tyrosine kinase inhibitor genistein (1010 , 108 ,106 , 105 M) were added to PGV cells for three days.
PGV cell numbers were not different at all concentrations of EGF, IGF-I and -II or insulin from controls(data not shown). Similarly, genistein had no effect oncell numbers (data not shown).

Fig. 3 Effects of PKC stimulators (PMA; 1012 M@105 M, andOAG; 108 M@104 M) on PGV cell number. PGV cells were cultured for 3 days, and cell numbers were counted with hemocytometer.
a, b and c denote signiﬁcant difference between groups ( p < 0:05).
Panel A, cell numbers with 106 M and 105 M PMA were signiﬁcantly higher than controls (10% HS alone). Panel B, cell numberswith 105 M and 104 M OAG were signiﬁcantly higher than controls.

PKA stimulators: PKA stimulators, forskolin (108 M@104 M) (þIBMX, 105 M), 8-bromo-cAMP (108 M@104 M) and dibutyryl cAMP (108 M@104 M)
were added to PGV cells and cell numbers were counted
after 3 days with hemocytometer.
All PKA stimulators had no effects on PGV cellnumbers (data not shown).
PKC stimulators and inhibitors: PKC stimulators,
PMA (1012 M@105 M) and OAG (108 M@104 M),
were added to PGV cells and cell numbers were counted
after 3 days. As shown in Fig. 3, both signiﬁcantly
stimulated PGV cell proliferation at concentrations ofgreater than 106 M PMA (Fig. 3-A) and at greaterthan 105 M OAG (Fig. 3-B).
PKC inhibitors, staurosporine (1012 M@108 M)
and calphostin C (1012 M@108 M) containing 10%
HS, were added to PGV cells. As shown in Fig. 4-A,
cell numbers with greater than 1010 M staurosporine
were signiﬁcantly less than controls (10% HS only).
Cell numbers with greater than 1010 M calphostin
C were also signiﬁcantly less than controls (data notshown). PMA (106 M) and staurosporine (1012 M@
108 M) were simultaneously added to PGV cells.
As shown in Fig. 4-B, 106 M PMA signiﬁcantly stimulated cell numbers compared with controls (10% HS
alone), while staurosporine signiﬁcantly inhibited cellnumbers in a dose-dependent manner, with a signiﬁcantdifference at greater than 1010 M staurosporine. Cell
numbers with 109 M and 108 M staurosporine were

Fig. 4 Effects of PKC inhibitor staurosporine (1012 M@108 M) on
proliferation of PGV cells. a, b, c and d denote signiﬁcant difference
between groups ( p < 0:05). Panel A, after 3 days of culture staurosporine signiﬁcantly inhibited cell numbers in a dose-dependent
manner, with signiﬁcant difference with 1010 M or greater compared
to controls (10% HS only). Panel B, PGV cells were cultured for 3
days with PMA (106 M) alone or in combination with increasing
concentrations of staurosporine (1012 M@108 M). PMA (106 M)
again signiﬁcantly stimulated PGV cell numbers, while staurosporine
dose-dependently inhibited PMA stimulation. PGV cells were notviable at 107 M staurosporine.

equal to controls. PGV cells were not viable at 107 M
staurosporine.
Calcium ionophore and calmodulin antagonists: Calcium ionophore A23187 (1010 M@105 M) and calmodulin antagonists, W5 (108 M@105 M) and W7 (108
M@105 M), were added to PGV cells and cell numbers
were counted after 3 days.

34

Fig. 5 Effects of PLC inhibitor U73122 (U; 1012 M@106 M) on
PGV cell number. a, b and c denote signiﬁcant difference between
groups ( p < 0:05). Panel A, after 3 days of culture PGV cell numberswith 107 M and 106 M U were signiﬁcantly less than controls (10%HS alone). Panel B, PGV cells were grown for 3 days with U (106 M)
alone or U (106 M) plus PMA (1012 M@106 M). U alone (106 M)
again signiﬁcantly inhibited PGV cell proliferation, while PMA(106 M) restored inhibition by U.

A23187, W5 and W7 had no effects on cell numbers(data not shown).
Phospholipase C inhibitor: PLC inhibitor U73122(1012 M@106 M) was added to PGV cells and cellnumbers were counted after 3 days. As shown in
Fig. 5-A, PGV cell numbers signiﬁcantly decreased with
107 M and 106 M U73122. When PMA (1012 M@
106 M) was simultaneously added to PGV cells with
106 M U73122, cell numbers with U73122 alone weresimilarly signiﬁcantly less than controls, while cellnumbers with 108 M PMA were slightly higher thanthose with U73122 alone, but the difference was not
signiﬁcant (Fig. 5-B). Cell numbers with 106 M PMA
were signiﬁcantly higher than those with U73122 alone
and compatible with controls (10% HS only).
Phosphatidic acid phosphatase inhibitor: Phosphatidic acid phosphatase (PAP) inhibitor propranolol(1012 M@105 M, PP) was added to PGV cells andcell numbers were counted after 3 days. PGV cellnumbers with PP decreased in a dose-dependent manner, with a signiﬁcant difference at 106 M and 105 M(Fig. 6-A).
When PMA (1012 M@106 M) was simultaneously
added to cells with PP (106 M), cell numbers increased
gradually according to the PMA doses shown in Fig.
6-B. PGV cell numbers with PP alone were similarly
signiﬁcantly less than controls (10% HS only), whilecell numbers with 1010 M PMA were signiﬁcantly
higher than those with PP alone, but still signiﬁcantly
lower than controls. PMA at 108 M or greater concentrations restored PGV cell proliferation that was
blocked by PP.

Lin M-T: Immortalized granulosa cell proliferation

Fig. 6 Effects of phosphatidic acid phosphatase inhibitor propranolol(PP; 1012 M@105 M) on PGV cell number. a, b and c denotesigniﬁcant difference between groups ( p < 0:05). Panel A, after 3
days of culture PGV cell numbers with 106 M and 105 M PP weresigniﬁcantly less than controls (10% HS alone). Panel B, PP alone(106 M) or PP (106 M) plus PMA (1012 M@106 M) were added to
PGV cell cultures. Cell numbers with PP alone were similarly signiﬁcantly less than controls, whereas those with 1010 M PMA plus PP(106 M) were signiﬁcantly higher than those with PP alone.

Discussion
Our results indicate that an immortalized porcine
granulosa cell line (PGV) has been successfully established using SV40 large T antigen. Although PGVcells were able to produce cAMP with response
to forskolin, PGV cells had no steroidogenic activity(Table 1) possibly due to the lack of transcription of
P450scc mRNA. However, this cell line signiﬁcantly
responded to stimulation by sera (Fig. 2) and also by
PKC (protein kinase C) stimulators (Fig. 3) in termsof cell proliferation. This prompted us to investigate
signaling pathways in PGV cells in terms of cell proliferation, with emphasis on the diacylglycerol (DAG)producing pathways.
Granulosa cell surface-speciﬁc antibodies were used
to immunologically stain PGV cells. PGV cells did
not stain with OG-1 antibody6 that reacts speciﬁcallyto human granulosa cells, whereas PGV cells stainedwith POG-1 antibody7 that reacts with porcine granulosa cells, theca interna cells and some stromal cells(unpublished observation, 1995). Since greater than
99.9% of aspirated cells from porcine ovarian follicles
in culture stained with oil red O (unpublished observation, 1985), they are steroid-producing cells (possibly
granulosa cells). These data may strongly suggest thatporcine granulosa cells are the origin of PGV cells.
Neither PKA stimulators nor growth factors, including insulin and tyrosine kinase inhibitor, inﬂuenced
PGV cell numbers. However, PKC stimulators signiﬁcantly increased PGV cell numbers (Fig. 3), while
PKC inhibitors signiﬁcantly reduced PGV cell proliferation that were stimulated by both sera and PKC stimulators (Figs. 4-A and 4-B, respectively). Inhibitor of

Keio J Med 2005; 54 (1): 29–38

PLC (phospholipase C), that catalyzes PIP2 (phosphatidylinositol 4, 5 bisphosphate) to DAG and IP3 (inositol 1, 4, 5-triphosphate), signiﬁcantly reduced PGV cellproliferation (Fig. 5-A). Moreover, inhibitor of phosphatidic acid phosphatase (PAP), that converts phosphatidic acid (PA) to DAG, also signiﬁcantly reduced
PGV cell numbers (Fig. 6-A). PMA restored PGV cellproliferation that was reduced by both PLC and PAP
inhibitors (Figs. 5-B and 6-B, respectively).
These data strongly suggest that both DAG and PKC
play an important role in PGV cell proliferation andthat DAG may be generated by PLC and also by PAP
in PGV cells. It is generally known that there are two
peaks of DAG production in many cells following agonist stimulation, the ﬁrst peak being transient and thelater sustained.8–10 The ﬁrst peak of DAG production
is generated by PLC from PIP2 , and the second is produced by PAP from PA. Moreover, the later sustained
DAG peak in cells activates PKC.11 PA is known to beconverted from phosphatidylcholine (PC) by PLD.8
Thus, our data also suggest that both PLC and PLD
pathways may work in PGV cells to produce two peaksof DAG from PIP2 and PC, respectively. Therefore, it
may be concluded that DAG, generated ﬁrst by PLC
and later by PLD, stimulates PKC in PGV cells similarly as in many other cells. The sustained PKC activity
may then stimulate PGV cell proliferation through yet
unknown mechanisms. Although PLA2 may be involved
in DAG and arachidonic acid generation in PGV cells,
this has not been conﬁrmed from our preliminary datausing the PLA2 inhibitor 4-bromophenacyl bromide(unpublished observation, 1998).
Further studies are necessary to clarity whether or
not FSH actually activates Ga, PLC, PLD, and then
PKC in PGV cells including mRNA expressions. DAG
production must also be determined temporally in PGVcells. Since FSH did not stimulate PGV cell proliferation in this study, it is inpossible to determine the timecourse of DAG, PLC, PLD or PKC activity in PGVcells after addition of FSH. This activity must be determined using other FSH-sensitive immortalized granulosa cells or primary granulosa cells following FSH
stimulation. In addition, the signaling pathways from
PKC to cyclins/cyclin-dependent kinases (cdk) mustalso be clariﬁed.
When FSH binds to its receptors, adenylate cyclase isactivated and generates cAMP from ATP (adenosine
triphosphate). The PKA pathway(s) does not regulate
cell proliferation nor steroidogenic activity in the PGVcells, while PKA systems do regulate production ofsteroids (progesterone and estrogen) in the primary
granulosa cells, as already shown by many investigators
including us.4 Although DAG concentrations have notyet been reported in the granulosa cells, DAG may
be generated in granulosa cells, as IP3 was detected

35

after LH stimulation.12,13 Using tissue cultures Peluso
et al.14 showed stimulation of granulosa cell proliferation by addition of PMA to rat granulosa cells, but thisstimulation was inhibited by 8-Br-cAMP. On the other
hand, cAMP promoted the proliferation of chicken
granulosa cells in culture.15 Sicinski et al. reported that
FSH stimulated granulosa cell numbers through acAMP-dependent pathway using granulosa cells fromcyclin D2-deﬁcient mice.16 However, other cyclin Ds(D1 and D3) were shown to be regulated by different
signaling pathways. Thus, there are still controversy
concerning whether the PKA or PKC pathways regulate cell proliferation of primary granulosa cells. However, there may be cross-talk between the A-kinase andC-kinase pathways.
IP3 , the other component catalyzed from PIP2 ,releases calcium ion from the endoplasmic reticulum.
Although it has been reported that FSH evokes an increase in intracellular free calcium ion concentrations insingle swine granulosa cells,17 calcium ion may not play
an important role in terms of PGV cell proliferation as
PGV cell numbers were inﬂuenced neither by calcium
ionophore nor by calmodulin antagonists in this study.
FBS stimulated PGV cell proliferation, whereas
neither EGF nor IGF stimulated PGV cell proliferation. As FBS contains many unknown factors, othergrowth factors or cytokines in FBS that have not yet
tested in this study may stimulate PGV cell proliferation. EGF has been shown to modulate FSH-induced
DNA synthesis in cultured granulosa cells.18 IGF-I
is also known to stimulate proliferation of granulosa
cells.19 Zhou et al. showed using IGF-I knockout micethat FSH does not affect IGF-I expression in granulosa
cells, but that IGF-I augments granulosa cell FSH
receptor expression.20 As PGV cell numbers were notinﬂuenced by EGF, IGF and genistein (tyrosine kinase
inhibitor), growth factor-tyrosine kinase-mitogenactivated protein kinase (MAPK) pathway(s) may not
function in the regulation of PGV cells. Our preliminary data suggest that MAPK activities were detected
5 to 10 minutes after FSH stimulation in cultured
primary granulosa cells (unpublished observation,1999). MAPK activity in granulosa cells after FSH
stimulation was also reported by Das et al.21 and Babu
et al.22 Das et al. showed that cAMP-directed pathways
by which FSH initiated granulosa cell differentiation
included activation of MAPK, while Babu et al. showedthat the cAMP independent growth promoting effectsof FSH were activated by calcium ion and MAPKdependent pathways. FSH receptor may have intrinsic
growth factor-like activity.23 Recently, phosphatidylinositol 3 (PI3) kinase-PKB pathway has been shown tobe present in granulosa cells24 and to be an essential
component of FSH-mediated granulosa cell differentiation.25 Our preliminary data demonstrate that the PI3

36kinase inhibitor wortmannin had no effect on PGV cellnumbers (unpublished observation, 1999). Thus among
the pathways tested in this study, with the exception of
PKC, other signaling pathways may not regulate cellproliferation in PGV cells.
The Jun family (c-Jun, Jun-B, and Jun-D), a basic
region leucine zipper family of transcription factors,
forms homodimers or heterodimers among Jun family
members and Fos family members (c-Fos, Fos-B, Fra-1,
and Fra-2). The dimer binds the consensus DNA
sequence (TGAC/GTCA) sites called AP-1 (activation proten-1) found in a variety of promoters.26,27
The PMA-responsive element, namely TRE (TPAresponsive element), is recognized by AP-1.28 AP-1 has
been shown to be involved in granulosa cell proliferation in response to tumor necrosis factor alpha.29
Serum response element (SRE) is also present in granulosa cells30 and SRE is involved in the expression ofc-fos by serum31 and is involved in the response to
PKC in many cells.32 Moreover, PKC is shown to
directly phosphorylate and activate Raf-1, part of the
MAPK kinase kinase (MAPKKK).33 Thus, there iscross-talk between known major signaling pathways
and possibly between other yet unknown signaling
pathways leading from FSH stimulation to granulosa
cell proliferation.
In PGV cells, progesterone was not detected eventhough it was stimulated by FSH or forskolin, whereas
primary granulosa cells responded to FSH and forskolin
in terms of progesterone production. One reason may
be the absence of P450scc mRNA (a rate-limiting enzyme of steroidogenesis) in PGV cells as evidenced by
Northern blotting. Retinoic acid was tested to determine whether it would restore steroidogenic activity,
but it failed to stimulate progesterone production in
PGV cells, although retinoic acid is able to block or reverse the carcinogenic process in various malignancies
including hematologic malignancies. It has also beenreported that in primary granulosa cells retinoic acidenhances the ability of FSH to induce LH-receptors andto stimulate the formation of cAMP and progesterone.34 Further studies are necessary to investigate why
the P450scc mRNA is not expressed in PGV cells, andalso whether PGV cells could restore steroidogenic
activity by means of transfection of P450scc gene to
PGV cells. Although PGV cells could not produce steroids, some cell lines including spontaneously established granulosa cell lines35,36 can secrete progesterone
and estrogen. These cells may be useful to investigate
the mechanisms of steroidogenesis in granulosa cells.
Immortalized granulosa cell lines were initiallyestablished by Amsterdam et al.37 followed by several
researchers. To immortalize granulosa cells, SV40
large T antigen alone,37,38 in combination with HaRAS oncogene,37 or in triple transfection with Ha-ras

Lin M-T: Immortalized granulosa cell proliferation

oncogene and FSH receptor expression plasmid,39 or
Kirsten-ras oncogene40 have previously been used totransfect rat, porcine or human granulosa cells. Immortalized granulosa cells have also been obtained fromtransgenic mouse ovaries.41 However, all these papers
focused on the mechanisms of steroidogenesis inimmortalized cells.
The intracellular signal transduction pathways inimmortalized granulosa cells regulating cell proliferation may differ from those of primary granulosa cells
inside follicles. However, it is not possible to replicate
in vitro the exact conditions of granulosa cells in follicles, as the granulosa cells luteinize spontaneously inculture and as cultured granulosa cells lose the capacity
to produce estrogens and to proliferate. Furthermore,
the steroidogenic and proliferative heterogeneity in
granulosa cells renders it difﬁcult to obtain pure, functionally deﬁned populations. Hence, cultured primary
granulosa cells are not appropriate to study the FSH
signaling pathways in granulosa cells inside the follicle,
although luteinized granulosa cells are often used tocharacterize granulosa cells in vitro. Therefore, it is advantageous to develop a homogeneous population of
granulosa cells. Thus, our immortalized granulosa cellline (PGV) can serve as a useful model to study thepotential mechanisms of granulosa cell proliferation.
Acknowledgements: The author would like to express sincere appreciation to Prof. Shiro Nozawa, Department of Obstetrics and Gynecology, School of Medicine, Keio University for his generous instruction and advice, and also to Dr. Kiyoo Tanabe, Department of
Obstetrics and Gynecology, Tokyo Denryoku Hospital for his kindassistance in performing this study. Sincere appreciation should also
go to Associate Professor Koichi Matsuo, Department of Microbiology, School of Medicine, Keio University for his kind instruction
to establish an immortalized granulosa cell line, Assistant Professor
Hiroshi Fujiwara, Department of Obstetrics and Gynecology, Facultyof Medicine, Kyoto University for immunostaining PGV cells with his
antibodies (OG-1 and POG-1), and to all members of the Keio HealthCounseling Center Gynecologic Endocrinology Study Group, Department of Obstetrics and Gynecology, School of Medicine, Keio
University for their kind assistance. The author is also grateful to
National Hormone and Pituitary Program, National Institutes of
Health, USA for providing human FSH and LH, and to Prof. Yoshiaki Fujii of Tohoku University for donating bovine P450scc cDNA,and to Fujisawa Pharmaceutical Co., Tokyo, Japan for providing
IGF-I and -II.

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