BIOLOGY OF REPRODUCTION 63, 15801585 (2000)
Effect of Cell-to-Cell Contact on In Vitro Deoxyribonucleic Acid Synthesis
and Apoptosis Responses of Bovine Granulosa Cells to Insulin-Like Growth Factor-I
and Epidermal Growth Factor1
A.M. Luciano,3 S. Modina,3 F. Gandolfi,3 A. Lauria,3 and D.T. Armstrong2,4
Istituto di Anatomia degli Animali Domestici,3 Universita di Milano, Milan, Italy
Reproductive Medicine Unit,4 Department of Obstetrics and Gynaecology, University of Adelaide,
The Queen Elizabeth Hospital, Woodville 5011, Australia
ABSTRACT
Follicle development is the result of a balanced ratio between
cell proliferation and cell death. Previous studies demonstrated
differential mitotic responses to insulin-like growth factor (IGF)-
I and epidermal growth factor (EGF) of cumulus cells (CC) and
mural granulosa cells (MGC). Because cell-to-cell contact seems
to modulate the occurrence of programmed cell death, the pre-
sent experiments investigated the role of cell association in me-
diating apoptosis and the mitogenic responses to these growth
factors of CC and MGC. Cumulus cells were cultured either as
intact cumulus-oocyte complexes (COC) or after dissociation
with EGTA 1 sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/
ml EGF, or both. Mural granulosa cells from the same follicles
were similarly cultured either as cell aggregates or as dissociated
cells. Synthesis of DNA was assessed by measurement of
[3H]thymidine incorporation during the last 6 h of a 24-h culture
in TCM199. Percentages of cells undergoing apoptosis were de-
termined immunohistochemically in intact COC and GC aggre-
gates, before and after dissociation as well as after the culture
period. Epidermal growth factor and IGF-I stimulated DNA syn-
thesis in both cell types; however, EGF inhibited the action of
IGF-I in intact COC but not in MGC. Compared to nondisso-
ciated cells, dissociation resulted in a reduction of the mitogenic
response of CC to both growth factors and of MGC to EGF.
Unlike the response of intact COC to combined treatment with
the two growth factors, dissociated CC displayed additive re-
sponses to the two growth factors in combination. Addition of
denuded oocytes to cultures of dissociated CC enhanced both
basal and growth factor-stimulated DNA synthesis but did not
restore the inhibitory effect of EGF on the IGF-I response char-
acteristic of intact COC. A significant proportion of intact MGC
aggregates underwent apoptosis after 24 h of culture, while no
increase of apoptotic cells was observed in intact COC. A dra-
matic increase in the percentage of apoptotic cells was observed
in both CC and MGC when cell-cell contact was interrupted,
and EGF and IGF-I were able to partially prevent its occurrence.
Taken together these data showed that CC and MGC exhibit
qualitatively and quantitatively different responses to IGF-I
when cultured in the presence of EGF both in terms of DNA
synthesis and onset of apoptosis. Moreover, the disruption of
1
This work was supported by the Italian Ministry of Agriculture Special
Project RAIZ and the Medical Research Council of Canada. D.T.A. was
supported by a RAISA Fellowship during sabbatical leave from the Uni-
versity of Western Ontario, Canada.
2
Correspondence: David T. Armstrong, Dept. of Obstetrics and Gynae-
cology, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville,
S.A., Australia 5011. FAX: 61 8 8222 7521;
e-mail: david.armstrong@adelaide.edu.au
Received: 27 January 2000.
First decision: 8 March 2000.
Accepted: 24 July 2000.
Q 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
1580
cell-cell contact was a major factor reducing cell proliferation
and inducing apoptosis among both subsets of GC.
apoptosis, cumulus cells, follicle, follicular development, granu-
losa cells, growth factors, ovary, pituitary hormones
INTRODUCTION
Cyclic ovarian follicular development is a complex pro-
cess that involves proliferation, differentiation, and death
of follicle cells [1]. Gonadotropins produced by the pitui-
tary gland have a central role in the regulation of these
processes. In addition, a wide range of paracrine and au-
tocrine factors produced in the ovary have also been pro-
posed as regulators of follicle growth.
During each estrous cycle, only one or a few follicles
mature and ovulate in mammals while the majority of fol-
licles ultimately undergo atresia. Follicular atresia seems to
be initiated with the death of the granulosa cells (GC) [1].
From in vivo and in vitro studies it has been demonstrated
that GC death is characterized by DNA fragmentation, sug-
gesting that GC die through an active process of pro-
grammed cell death or apoptosis [2, 3]. It has recently been
suggested that cell-cell adhesion may affect the suscepti-
bility of GC to apoptosis [4]. Moreover, recent studies dem-
onstrated the possible role of gap and tight junctions in
preventing the occurrence of GC apoptosis [5, 6].
With the emerging concepts of how GC die, it is im-
portant to appreciate the considerable heterogeneity in the
somatic cell compartments of the follicle. As follicle de-
velopment progresses from the preantral to the preovulatory
stages, a regional differentiation of GC function occurs
from the peripheral (mural) GC (MGC) to those closest to
the oocyte (cumulus and corona radiata cells, CC). Differ-
ences among GC subsets may be influenced both by prox-
imity to blood supply (restricted to the thecal layer of the
follicle) and by diffusion gradients of paracrine factors se-
creted into the follicular fluid by different follicle cell types
including thecal cells, GC themselves, as well as the oo-
cyte.
Growth factors influence follicle growth, in part, by
stimulating DNA synthesis and by modulating the onset of
apoptosis in GC. Growth factors of particular importance
in GC regulation include insulin-like growth factor (IGF)-
I and epidermal growth factor (EGF). In addition to acting
as a potent GC mitogen, IGF-I influences both proliferation
and differentiation responses of GC to gonadotropins [7].
Responses of GC to EGF include proliferation of MGC and
mucification of CC [8]. Both IGF-I and EGF exert inhibi-
tory influences on apoptosis in various cell types including
GC [9, 10].
Previous studies of responses of bovine GC subsets to
hormones and growth factors have shown a differential re-
sponse of MGC and CC to IGF-I and EGF in terms of both
 CELL-CELL CONTACT AND GRANULOSA CELL FUNCTIONS
DNA synthesis and progesterone secretion [11, 12]. Mural
GC and intact CC-oocyte complexes (COC) each respond-
ed to IGF-I and to EGF with increased DNA synthesis.
However, the two factors in combination elicited additive
responses in mural cells, whereas in the presence of EGF,
the stimulatory effect of IGF-I on COC was markedly at-
tenuated or absent. The present study was undertaken to
determine if these differential responses to IGF-I and EGF
could be attributed to differences in the onset of apoptosis
between the two GC subtypes and to evaluate the role of
cell-cell contact in the mitogenic responses of GC to these
growth factors in both cell populations. In addition, the re-
spective roles of cell-cell contact and of factor(s) secreted
by the oocyte in determining the characteristic mitogenic
responses of the COC to IGF-I and EGF was assessed.
MATERIALS AND METHODS
Cell Isolation and Culture
All reagents were purchased from Sigma Chemical Co.
(St. Louis, MO), unless otherwise indicated.
Ovaries were obtained from a local slaughterhouse and
held al 32368C in PBS with antibiotic-antimycotic solution
for up to 2 h during transport to the laboratory, pending
follicular aspiration. Follicles 25 mm in diameter were as-
pirated with an 18-gauge needle on a 10-ml syringe con-
taining a small volume of aspiration medium, M-199 con-
taining Hepes 25 mM and BSA 0.4% (M-199A). Cumulus-
oocyte complexes and MGC aggregates were allowed to
settle from the follicular aspirates and COC removed under
microscopic examination. The isolated COC were washed
through three rinses of aspiration medium and three times
in bicarbonate-buffered medium M-199 without serum (M-
199C). Granulosa cell aggregates were similarly resuspend-
ed and washed sequentially twice in M-199A and once in
M-199C. Each time MGC aggregates were allowed to settle
for 5 min so that single cells were removed from the sus-
pension.
Cumulus cells and MGC were cultured either as intact
COC and MGC aggregates or as dispersed cell prepara-
tions. Dispersion of both COC and mural cells was per-
formed by the hypertonic method developed by Campbell
[13], with slight modifications. Briefly, COC or GC aggre-
gates were suspended in M-199 containing 0.2% BSA, 9.1
mM EGTA at pH 7.4, and incubated at 378C for 5 min.
The supernatant was then removed and cells transferred to
M-199 containing 0.2% BSA, 2.1 mM EGTA, and 0.5 M
sucrose at pH 7.4 solution for an additional 10-min incu-
bation. After dissociation, CC and MGC were washed twice
in bicarbonate-buffered M-199C, counted with a hemocy-
tometer, and viability assessed by trypan blue exclusion.
Analogous procedures were applied to groups of 10 COC
to assess the number of cells in each complex. The average
number of CC was 3.2 3 103/COC. This mean value was
multiplied by the number of COC per well to provide an
estimate of the number of cumulus cells as intact COC per
well.
Cultures of intact and dissociated cells of both types (ex-
periments 1 and 3) were carried out in 0.25 ml M-199C in
96-well flat-bottomed microwell tissue culture plates. Cu-
mulus cells were cultured in wells containing either 810
intact COC estimated to contain 2.53.0 3 104 CC, or an
equivalent number of dispersed CC, in 0.25 ml M-199C.
Mural GC were cultured at 2.5 3 105 cells per well, either
as cell aggregates or dissociated cells.
To evaluate the effects of oocyte-secreted factor(s) in
1581
FIG. 1. Synthesis of DNA in CC: influence of cell association and growth
factors. Data from three pooled experiments analyzed by ANOVA fol-
lowed by Student-Newman-Keuls test. Different superscripts indicate sta-
tistical difference P , 0.001.
determining mitogenic response of CC to growth factors
(experiment 2), the oocytes released from the dissociated
COC were recovered under microscopic examination and
vortexed for 2 min in 2 ml M-199 in a 15-ml centrifuge
tube to remove any remaining adherent CC. The resulting
denuded oocytes (DO) were pooled for coculture with CC.
Dissociated CC were cultured alone or in coculture with 40
DO per well, in a reduced volume (0.125 ml) of M-199C
containing 30 000 CC per well.
All cultures were performed in duplicate or triplicate
wells per treatment per experiment, with experiments rep-
licated three times. Treatments comprised no growth factor
(basal), 50 ng/ml recombinant human IGF-I (Boehringer
Mannheim, Roche Diagnostics S.p.A., Milan, Italy), 5 ng/
ml recombinant murine EGF, or the two growth factors in
combination. Cell cultures were incubated in a humidified
incubator for 24 h at 38.58C in 5% CO295% air.
Assessment of DNA Synthesis
After 18 h of culture 0.1 mCi [3H]thymidine was added
to each well, and cultures were continued for 6 h to permit
labeling of DNA as an index of cell proliferation. Mural
GC and CC were harvested and collected by vacuum man-
ifold system (Millipore S.p.A., Milan, Italy), or with a Tom-
tec Cell Harvester 96 (Tomtec, Hamden, CT) onto glass
fiber filters and then washed to remove soluble radioactiv-
ity. Incorporated radioactivity was determined by liquid
scintillation counting of the cells collected on glass fiber
filters.
Assessment of Apoptosis In Situ
Cells undergoing apoptosis were identified with the In
Situ Apoptotic Cell Death Detection Kit (Boehringer Mann-
heim). The same concentration of cells used in DNA syn-
thesis determination were plated before and after dissocia-
tion in an eight-chamber Lab-Tek slide (Nunc, Life Tech-
nologies, Milan, Italy) and cultured at 38.58C in an atmo-
sphere of 5% CO2 in air, for 24 h, in the presence of the
same combination of growth factors used before. At the
time of harvesting (Time 0 h) and at the end of the culture
period (Time 24 h), medium was removed, and cells were
washed once in PBS and air dried at 378C. Cells were fixed
with 4% paraformaldehyde in PBS and stained using re-
agents and instructions provided with the kit. The presence
of DNA fragments was detected by the TUNEL technique.
This is a very sensitive technique that preferentially labels
1582 LUCIANO ET AL.

FIG. 2. Synthesis of DNA in MGC: influence of cell association and

growth factors. Data from three pooled experiments analyzed by ANOVA
followed by Student-Newman-Keuls test. Different superscripts indicate
statistical difference P , 0.001.

apoptotic cells in comparison to necrotic cells, thereby dis-

criminating apoptosis from necrosis and from primary
DNA strand breaks. Cells were incubated 60 min at 378C
with the TUNEL mixture, allowing fluorescein-dUTP to la-
bel DNA strand breaks. The cells were then washed once
with PBS and observed under epifluorescence and bright-
field optics (Nikon Diaphot, Tokyo, Japan). Between 200
and 300 cells were individually observed for each treatment
and used to calculate the percentage of cells with apoptotic
nuclei.

Statistical Analysis
All experiments were repeated at least three times, with
two or three replicate wells per treatment in each experi-
ment. The data were analyzed statistically by multivariate
analysis of variance using SPSS, followed by Student-New-
man-Keuls multiple range test for comparison of individual
means. Significance of differences were inferred at P ,
0.05.

RESULTS
Experiment 1: Effect of Cell Dissociation on DNA
Synthesis in Response to Growth Factors
The effects of EGF and IGF-I on [3H]thymidine incor-
poration in CC and MGC are presented in Figure 1 for CC
and Figure 2 for MGC.
When added to cultures of intact COC or MGC aggre-
gates, IGF-I resulted in a highly significant (P , 0.001)
increase in [3H]thymidine incorporation, while only mar-
ginal, statistically insignificant increases occurred in both
GC subtypes in response to EGF. Incorporation in CC was
substantially higher than in MGC (P , 0.001) under both
basal and IGF-I-stimulated conditions. Moreover, a diver-
gence of responses of the two cell types was observed with
the combination of EGF and IGF-I: MGC responded in an
additive manner, whereas EGF was highly inhibitory to the
proliferative response of intact COC to IGF-I.
FIG. 3. Morphology (brightfield optics) and in situ cell death detection
(epifluorescence) in aggregated MGC at Time 0 h (A, B), after 24 h of
culture (C, D), in dispersed MGC at Time 0 h (E, F), and after 24 h of
culture (G, H). Cells undergoing apoptosis identified with the In Situ Ap-
optotic Cell Death Detection Kit (Boehringer Mannheim). Magnification
3100.
In the absence of growth factors (basal conditions), mean
6 SEM [3H]thymidine incorporation in undissociated CC
and MGC aggregates were 791 6 135 and 104 6 18 dpm/
103 cells, respectively, compared to 120 6 17 and 65 6 6
dpm/103 cells, in dissociated CC and MGC. Cell dissocia-
tion resulted in 6.5-fold and 2-fold reductions in
[3H]thymidine incorporation in CC and MGC, respectively,
under basal conditions and significantly reduced DNA syn-
thesis in both cell types in the presence of growth factors

TABLE 1. Effect of EGF and IGF-I on DNA synthesis in dissociated CC cultured alone and in coculture with DO.a

Cell types Control EGF IGF-I EGF 1 IGF-I

Dissociated CC 32 6 4 A 56 6 9 B 90 6 18 C 148 6 21 D
Dissociated CC 1 DO 60 6 4 B 85 6 8 C 135 6 9 D 160 6 14 D
aData are mean 6 SEM dpm [3H]thymidine incorporation per 1000 CC. Different letters indicate statistical difference (within rows, P , 0.01, Student-
Newman-Keuls test; within columns, P , 0.01, multivariate ANOVA).
 CELL-CELL CONTACT AND GRANULOSA CELL FUNCTIONS 1583

TABLE 2. Effect of EGF and IGF-I on the percentage of cumulus undergoing apoptosis at Time 0 and after 24 h of culture.a

Control EGF IGF-I EGF 1 IGF-I

Time 0 h 1.2 6 0.4 A
Undissociated COC 1.6 6 0.4 A 0.9 6 0.6 A 1.2 6 0.6 A 2.5 6 0.7 A
Dissociated CC 85.3 6 2.2 D 69.3 6 1.4 C 70.7 6 3.3 C 55.4 6 5.1 B
aData are expressed as percentages of apoptotic cells calculated after the observation of 200300 cells for each treatment. Different letters within rows
or columns indicate statistical difference (P , 0.001, Student-Newman-Keuls test).

(P , 0.001). Dissociation of CC also altered the pattern of
response to IGF-I in combination with EGF; whereas EGF
inhibited the response of intact COC to IGF-I, the two
growth factors together elicited approximately additive re-
sponses in dissociated CC.
Experiment 2: Effect of Coculture of Dispersed CC
with DO on DNA Synthesis
The effects of coculture of dispersed CC with DO are
summarized in Table 1. As in experiment 1, both EGF and
IGF-I by themselves significantly increased DNA synthesis
in dissociated CC, and together elicited approximately ad-
ditive responses. Coculture with DO resulted in a twofold
increase in [3H]thymidine incorporation in dispersed CC in
the absence of added growth factors and further increased
the responses to EGF or IGF-I alone, with no significant
interaction between growth factors and cell types. Thus,
DO were unable to restore the inhibitory effect of EGF on
the IGF-I response characteristic of intact COC, indicating
that the effect of cell dissociation in altering the respective
responses of COC to combined treatment with the two
growth factors could not be attributed solely to absence of
oocyte-secreted factors.
Experiment 3: Effect of Growth Factors and Cell
Dissociation on Apoptosis
Figure 3 shows immunohistochemical detection of apo-
ptotic cells of aggregated and dispersed MGC.
Results of apoptotic cell counts in intact aggregates and
dispersed cell preparations of CC and MGC at time of iso-
lation from follicles (0 h) and after 24 h of culture in dif-
ferent treatments are presented in Tables 2 and 3, respec-
tively.
At Time 0 h, between 12% of cells showed DNA frag-
mentation characteristic of apoptosis in both intact and dis-
persed CC and between 46% in both intact and dispersed
MGC. A significant proportion of aggregated MGC (up to
23.0 6 2.1%) underwent apoptosis after 24 h of culture
while no increase in apoptotic cells was observed in the
intact CC. Growth factors were able to partially prevent the
occurrence of apoptosis in intact MGC.
A dramatic increase in the percentage of apoptotic cells
was observed in both CC and MGC when cell-to-cell con-
tact was interrupted by cell dispersal. Both EGF and IGF-
I were able to reduce significantly the extent of apoptosis
in both subsets of dispersed cells. Moreover when added in
combination, these growth factors were able to suppress
apoptosis of dispersed CC and GC to a level significantly
below that resulting from either growth factor by itself.
DISCUSSION
The results of the present study confirm the occurrence
of apoptosis in bovine GC during in vitro culture and show,
for the first time, that CC do not undergo apoptosis after
24 h of culture as intact COC in defined media. In addition,
this is the first report that eliminating cell-cell contact alters
the proliferative responses of CC to growth factors in a
manner different from that of MGC.
Growth factors play fundamental roles in regulating the
proliferation and differentiation of follicle constituents by
direct actions as well as by influencing actions of gonad-
otrophic hormones [7, 14]. Growth factors seem to modu-
late the balance between GC survival and cell death. More
than 99% of ovarian follicles undergo atresia during repro-
ductive life. It has been shown that apoptotic cell death of
GC is the molecular mechanism underlying follicle atresia
[1, 15, 16]. An inhibitory effect of EGF on apoptosis in
cultured rat GC has been reported [15, 17], an effect that
is suggested to be mediated, at least in part, by enhanced
synthesis of progesterone [10]. A direct inhibitory action
of IGF-I on apoptosis in cultured pig GC has recently been
reported [18].
In the present studies, the disruption of cell-cell contact
was the main factor reducing cell proliferation capability of
both MGC and CC, and inducing apoptosis among both
GC subsets. This is consistent with the observation that the
disruption of extracellular matrix (ECM) and the inhibition
of intercellular contact are important factors inducing ap-
optosis in others cell types [19, 20]. The involvement of
cell-cell contact is further supported by the observation that
junctional complexes are reduced within atretic follicles
[21]. In healthy ovarian follicles, GC are connected by gap
junctions, and these gap junctional complexes coordinate
functional responses between GC and between GC and the
oocyte [21, 22]. Moreover, recent findings suggested a pos-
sible involvement of the adhesion junction protein N-cad-
herin in triggering a signal transduction cascade that ulti-
mately inhibits apoptosis [5, 6].
Variations in IGF-binding protein (IGFBP) secretion by
GC occur in follicles in different physiological states. High
follicular fluid levels of IGFBP4 are characteristic of atretic
follicles, and direct correlations between the increase in
IGFBPs and the progression from healthy to atretic follicles

TABLE 3. Effect of EGF and IGF-I on the percentage of MGC undergoing apoptosis at Time 0 and after 24 h of culture.a

Control EGF IGF-I EGF 1 IGF-I

Time 0 h 5.6 6 1.3 A
MGC undissociated 23.0 6 2.1 C 14.1 6 1.7 B 14.9 6 2.4 B 14.2 6 1.7 B
Dissociated MGC 96.4 6 1.0 D 59.2 6 7.4 C 66.5 6 6.7 C 47.7 6 3.4 B
aData are expressed as percentages of apoptotic cells calculated after the observation of 200300 cells for each treatment. Different letters within rows
or columns indicate statistical difference (P , 0.001, Student-Newman-Keuls test).
1584 LUCIANO ET AL.
[23] have been reported. Secretion of IGFBPs by GC is
influenced by gonadotropins and growth factors, with FSH
stimulating IGFBP3 production and altering the ratio be-
tween different IGFBPs in sheep and bovine GC [24, 25].
Inhibitory effects of IGFBPs on both proliferative and se-
cretory responses of rat GC have been reported previously
[26], and FSH at low doses stimulates IGFBP3 production
by rat and bovine GC [25, 27].
The IGFBPs are important components of ECM that can
have major influences on cell responses to IGF-I [27]. The
IGFBPs vary in their ability to influence bioactivity of IGF,
and this in turn can be influenced by the state of the ECM.
Insulin-like growth factor-BP3 is an effective inhibitor of
IGF-I actions in many cell systems by binding available
IGF-I, thereby limiting its availability to cell receptors that
mediate its actions. On the other hand, IGFBP5 can en-
hance the activity of IGF-I through more effective delivery
to IGF receptors on the cell surface [28]. Both FSH and
EGF markedly alter the composition and structure of the
ECM in intact CC as a consequence of increased secretion
of hyaluronic acid and glycosaminoglycans that contribute
to the mucification of CC mass in response to these agents
[8, 29]. Alterations in the composition of the ECM of COC
accompanying mucification could conceivably influence its
ability to bind IGFBPs, thus modifying the proliferative and
antiapoptotic responses of CC to IGF-I. Recently reported
differences between MGC and COC in the pattern of
IGFBP secretion and retention in the ECM in response to
mucifiying treatment offer support for this possibility [25].
Follicle-stimulating hormone by itself stimulated IGFBP3
secretion by OCC but not MGC, an effect that was aug-
mented by IGF-I. Both cell types also secreted IGFBP5, a
greater proportion of which was retained in the ECM of
COC than of MGC, and combined treatment with FSH plus
IGF-I increased the retention of IGFBP5 in the ECM of
COC but not of MGC. [25].
In the present study, the previously reported differences
between CC and MCG in DNA synthesis induced by IGF-
I and EGF were abolished by cell dissociation. Differences
in ECM composition in the two intrafollicular compart-
ments may partially explain these functional differences.
Loss of ECM as a result of dissociation of CC may have
reduced or eliminated modulating effects of ECM compo-
nents including bound IGFBPs associated with CC. The
resulting absence of ECM-associated IGFBP5 may have al-
tered the CC response to IGF-I, eliminating the inhibitory
effect of EGF on DNA synthesis seen in intact CC. On the
contrary, because the ECM of MGC does not undergo such
changes in response to EGF, the relative responses of MGC
to IGF-I are not altered either by EGF or by cell dissoci-
ation of the GC aggregates.
An additional possible contributing factor to the differ-
ences in responses of MGC and CC to growth factors and
to cell dissociation may be oocyte-secreted factors. Consid-
erable attention has focussed recently on regulatory actions
of oocyte-secreted factors in a variety of differentiated re-
sponses of CC, including FSH- and EGF-induced cumulus
expansion [29] and granulosa cell proliferation, based on
studies involving removal of oocytes from COC (oocytec-
tomy) and supplementation of granulosa cultures with oo-
cyte-conditioned media [30]. Oocytectomy of bovine COC
was reported recently to decrease DNA synthesis in CC and
to alter the pattern of their mitogenic response to IGF-I in
combination with FSH, such that the two agents stimulated
DNA synthesis in an additive manner similar to that char-
acteristic of MGC. Coculture of oocytectomized complexes
with DO increased DNA synthesis and restored their rela-
tive responses to IGF-I plus FSH to a pattern similar to that
observed with intact COC [31, 32], thus supporting a role
of oocyte-secreted factor(s) in influencing the response of
CC to IGF-I. In contrast, coculture with DO in the present
study increased DNA synthesis in dissociated CC irrespec-
tive of the presence of both growth factors, whether alone
or in combination. These findings indicate that the differ-
ence in response of oocytectomized versus dissociated CC
complexes to IGF is attributable to loss of cell-to-cell con-
tact and/or ECM components in the latter cell preparation,
rather than to lack of oocyte-secreted factors. Further re-
search is required to elucidate the complex interactions be-
tween oocyte-secreted factors, ECM with its associated
IGF-binding proteins, and direct cell-to-cell contact in de-
termining the differential phenotypes of different GC sub-
sets and their responses to hormonal and paracrine signals
within the follicle throughout its growth and maturation.
In summary, the present results confirm our previous
findings of different mitogenic responses of the two follic-
ular GC subsets to growth factors and extend the findings
to include measurements of apoptosis in cells cultured un-
der the same conditions. Disruption of cell-cell contact by
cell dissociation decreases DNA synthesis and alters the
response of CC to combined treatment with EGF and IGF-
I from mutual antagonism charactistic of intact COC to an
additive response. Coculture of dissociated CC with DO
increases DNA synthesis and response to each of the
growth factors alone in an additive manner but does not
restore their mutual antagonistic effects when added in
combination. Unlike MGC, CC do not undergo apoptosis
if cultured as intact COC. Dissociation of both MGC and
COC triggers apoptosis in both cell subsets, and both IGF-
I and EGF are able to partially prevent the occurrence of
apoptosis in both cell subsets.
ACKNOWLEDGMENTS
We gratefully acknowledge the expert technical assistance of Ms. Les-
ley Ritter and Ms. Rebecca Thomas and the helpful suggestions of Dr.
Robert Gilchrist concerning oocyte-secreted factors.
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