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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 cell-cell contact was a major factor reducing cell proliferation

and inducing apoptosis among both subsets of GC.
Follicle development is the result of a balanced ratio between
cell proliferation and cell death. Previous studies demonstrated apoptosis, cumulus cells, follicle, follicular development, granu-
differential mitotic responses to insulin-like growth factor (IGF)- losa cells, growth factors, ovary, pituitary hormones
I and epidermal growth factor (EGF) of cumulus cells (CC) and
mural granulosa cells (MGC). Because cell-to-cell contact seems INTRODUCTION
to modulate the occurrence of programmed cell death, the pre- Cyclic ovarian follicular development is a complex pro-
sent experiments investigated the role of cell association in me- cess that involves proliferation, differentiation, and death
diating apoptosis and the mitogenic responses to these growth of follicle cells [1]. Gonadotropins produced by the pitui-
factors of CC and MGC. Cumulus cells were cultured either as tary gland have a central role in the regulation of these
intact cumulus-oocyte complexes (COC) or after dissociation
processes. In addition, a wide range of paracrine and au-
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
tocrine factors produced in the ovary have also been pro-
were similarly cultured either as cell aggregates or as dissociated posed as regulators of follicle growth.
cells. Synthesis of DNA was assessed by measurement of During each estrous cycle, only one or a few follicles
[3H]thymidine incorporation during the last 6 h of a 24-h culture mature and ovulate in mammals while the majority of fol-
in TCM199. Percentages of cells undergoing apoptosis were de- licles ultimately undergo atresia. Follicular atresia seems to
termined immunohistochemically in intact COC and GC aggre- be initiated with the death of the granulosa cells (GC) [1].
gates, before and after dissociation as well as after the culture From in vivo and in vitro studies it has been demonstrated
period. Epidermal growth factor and IGF-I stimulated DNA syn- that GC death is characterized by DNA fragmentation, sug-
thesis in both cell types; however, EGF inhibited the action of gesting that GC die through an active process of pro-
IGF-I in intact COC but not in MGC. Compared to nondisso- grammed cell death or apoptosis [2, 3]. It has recently been
ciated cells, dissociation resulted in a reduction of the mitogenic suggested that cell-cell adhesion may affect the suscepti-
response of CC to both growth factors and of MGC to EGF. bility of GC to apoptosis [4]. Moreover, recent studies dem-
Unlike the response of intact COC to combined treatment with onstrated the possible role of gap and tight junctions in
the two growth factors, dissociated CC displayed additive re- preventing the occurrence of GC apoptosis [5, 6].
sponses to the two growth factors in combination. Addition of With the emerging concepts of how GC die, it is im-
denuded oocytes to cultures of dissociated CC enhanced both portant to appreciate the considerable heterogeneity in the
basal and growth factor-stimulated DNA synthesis but did not somatic cell compartments of the follicle. As follicle de-
restore the inhibitory effect of EGF on the IGF-I response char- velopment progresses from the preantral to the preovulatory
acteristic of intact COC. A significant proportion of intact MGC stages, a regional differentiation of GC function occurs
aggregates underwent apoptosis after 24 h of culture, while no from the peripheral (mural) GC (MGC) to those closest to
increase of apoptotic cells was observed in intact COC. A dra-
matic increase in the percentage of apoptotic cells was observed
the oocyte (cumulus and corona radiata cells, CC). Differ-
in both CC and MGC when cell-cell contact was interrupted,
ences among GC subsets may be influenced both by prox-
and EGF and IGF-I were able to partially prevent its occurrence. imity to blood supply (restricted to the thecal layer of the
Taken together these data showed that CC and MGC exhibit follicle) and by diffusion gradients of paracrine factors se-
qualitatively and quantitatively different responses to IGF-I creted into the follicular fluid by different follicle cell types
when cultured in the presence of EGF both in terms of DNA including thecal cells, GC themselves, as well as the oo-
synthesis and onset of apoptosis. Moreover, the disruption of cyte.
Growth factors influence follicle growth, in part, by
This work was supported by the Italian Ministry of Agriculture Special
stimulating DNA synthesis and by modulating the onset of
Project RAIZ and the Medical Research Council of Canada. D.T.A. was apoptosis in GC. Growth factors of particular importance
supported by a RAISA Fellowship during sabbatical leave from the Uni- in GC regulation include insulin-like growth factor (IGF)-
versity of Western Ontario, Canada. I and epidermal growth factor (EGF). In addition to acting
Correspondence: David T. Armstrong, Dept. of Obstetrics and Gynae- as a potent GC mitogen, IGF-I influences both proliferation
cology, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville, and differentiation responses of GC to gonadotropins [7].
S.A., Australia 5011. FAX: 61 8 8222 7521;
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
Received: 27 January 2000.
First decision: 8 March 2000.
GC [9, 10].
Accepted: 24 July 2000. Previous studies of responses of bovine GC subsets to
Q 2000 by the Society for the Study of Reproduction, Inc. hormones and growth factors have shown a differential re-
ISSN: 0006-3363. sponse of MGC and CC to IGF-I and EGF in terms of both

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 FIG. 1. Synthesis of DNA in CC: influence of cell association and growth
responses of the COC to IGF-I and EGF was assessed. factors. Data from three pooled experiments analyzed by ANOVA fol-
lowed by Student-Newman-Keuls test. Different superscripts indicate sta-
MATERIALS AND METHODS tistical difference P , 0.001.

Cell Isolation and Culture

All reagents were purchased from Sigma Chemical Co. determining mitogenic response of CC to growth factors
(St. Louis, MO), unless otherwise indicated. (experiment 2), the oocytes released from the dissociated
Ovaries were obtained from a local slaughterhouse and COC were recovered under microscopic examination and
held al 32368C in PBS with antibiotic-antimycotic solution vortexed for 2 min in 2 ml M-199 in a 15-ml centrifuge
for up to 2 h during transport to the laboratory, pending tube to remove any remaining adherent CC. The resulting
follicular aspiration. Follicles 25 mm in diameter were as- denuded oocytes (DO) were pooled for coculture with CC.
pirated with an 18-gauge needle on a 10-ml syringe con- Dissociated CC were cultured alone or in coculture with 40
taining a small volume of aspiration medium, M-199 con- DO per well, in a reduced volume (0.125 ml) of M-199C
taining Hepes 25 mM and BSA 0.4% (M-199A). Cumulus- containing 30 000 CC per well.
oocyte complexes and MGC aggregates were allowed to All cultures were performed in duplicate or triplicate
settle from the follicular aspirates and COC removed under wells per treatment per experiment, with experiments rep-
microscopic examination. The isolated COC were washed licated three times. Treatments comprised no growth factor
through three rinses of aspiration medium and three times (basal), 50 ng/ml recombinant human IGF-I (Boehringer
in bicarbonate-buffered medium M-199 without serum (M- Mannheim, Roche Diagnostics S.p.A., Milan, Italy), 5 ng/
199C). Granulosa cell aggregates were similarly resuspend- ml recombinant murine EGF, or the two growth factors in
ed and washed sequentially twice in M-199A and once in combination. Cell cultures were incubated in a humidified
M-199C. Each time MGC aggregates were allowed to settle incubator for 24 h at 38.58C in 5% CO295% air.
for 5 min so that single cells were removed from the sus-
pension. Assessment of DNA Synthesis
Cumulus cells and MGC were cultured either as intact After 18 h of culture 0.1 mCi [3H]thymidine was added
COC and MGC aggregates or as dispersed cell prepara- to each well, and cultures were continued for 6 h to permit
tions. Dispersion of both COC and mural cells was per- labeling of DNA as an index of cell proliferation. Mural
formed by the hypertonic method developed by Campbell GC and CC were harvested and collected by vacuum man-
[13], with slight modifications. Briefly, COC or GC aggre- ifold system (Millipore S.p.A., Milan, Italy), or with a Tom-
gates were suspended in M-199 containing 0.2% BSA, 9.1 tec Cell Harvester 96 (Tomtec, Hamden, CT) onto glass
mM EGTA at pH 7.4, and incubated at 378C for 5 min. fiber filters and then washed to remove soluble radioactiv-
The supernatant was then removed and cells transferred to ity. Incorporated radioactivity was determined by liquid
M-199 containing 0.2% BSA, 2.1 mM EGTA, and 0.5 M scintillation counting of the cells collected on glass fiber
sucrose at pH 7.4 solution for an additional 10-min incu- filters.
bation. After dissociation, CC and MGC were washed twice
in bicarbonate-buffered M-199C, counted with a hemocy- Assessment of Apoptosis In Situ
tometer, and viability assessed by trypan blue exclusion.
Analogous procedures were applied to groups of 10 COC Cells undergoing apoptosis were identified with the In
to assess the number of cells in each complex. The average Situ Apoptotic Cell Death Detection Kit (Boehringer Mann-
number of CC was 3.2 3 103/COC. This mean value was heim). The same concentration of cells used in DNA syn-
multiplied by the number of COC per well to provide an thesis determination were plated before and after dissocia-
estimate of the number of cumulus cells as intact COC per tion in an eight-chamber Lab-Tek slide (Nunc, Life Tech-
well. nologies, Milan, Italy) and cultured at 38.58C in an atmo-
Cultures of intact and dissociated cells of both types (ex- sphere of 5% CO2 in air, for 24 h, in the presence of the
periments 1 and 3) were carried out in 0.25 ml M-199C in same combination of growth factors used before. At the
96-well flat-bottomed microwell tissue culture plates. Cu- time of harvesting (Time 0 h) and at the end of the culture
mulus cells were cultured in wells containing either 810 period (Time 24 h), medium was removed, and cells were
intact COC estimated to contain 2.53.0 3 104 CC, or an washed once in PBS and air dried at 378C. Cells were fixed
equivalent number of dispersed CC, in 0.25 ml M-199C. with 4% paraformaldehyde in PBS and stained using re-
Mural GC were cultured at 2.5 3 105 cells per well, either agents and instructions provided with the kit. The presence
as cell aggregates or dissociated cells. of DNA fragments was detected by the TUNEL technique.
To evaluate the effects of oocyte-secreted factor(s) in This is a very sensitive technique that preferentially labels

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

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 ,

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

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


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


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).

[23] have been reported. Secretion of IGFBPs by GC is with DO increased DNA synthesis and restored their rela-
influenced by gonadotropins and growth factors, with FSH tive responses to IGF-I plus FSH to a pattern similar to that
stimulating IGFBP3 production and altering the ratio be- observed with intact COC [31, 32], thus supporting a role
tween different IGFBPs in sheep and bovine GC [24, 25]. of oocyte-secreted factor(s) in influencing the response of
Inhibitory effects of IGFBPs on both proliferative and se- CC to IGF-I. In contrast, coculture with DO in the present
cretory responses of rat GC have been reported previously study increased DNA synthesis in dissociated CC irrespec-
[26], and FSH at low doses stimulates IGFBP3 production tive of the presence of both growth factors, whether alone
by rat and bovine GC [25, 27]. or in combination. These findings indicate that the differ-
The IGFBPs are important components of ECM that can ence in response of oocytectomized versus dissociated CC
have major influences on cell responses to IGF-I [27]. The complexes to IGF is attributable to loss of cell-to-cell con-
IGFBPs vary in their ability to influence bioactivity of IGF, tact and/or ECM components in the latter cell preparation,
and this in turn can be influenced by the state of the ECM. rather than to lack of oocyte-secreted factors. Further re-
Insulin-like growth factor-BP3 is an effective inhibitor of search is required to elucidate the complex interactions be-
IGF-I actions in many cell systems by binding available tween oocyte-secreted factors, ECM with its associated
IGF-I, thereby limiting its availability to cell receptors that IGF-binding proteins, and direct cell-to-cell contact in de-
mediate its actions. On the other hand, IGFBP5 can en- termining the differential phenotypes of different GC sub-
hance the activity of IGF-I through more effective delivery sets and their responses to hormonal and paracrine signals
to IGF receptors on the cell surface [28]. Both FSH and within the follicle throughout its growth and maturation.
EGF markedly alter the composition and structure of the In summary, the present results confirm our previous
ECM in intact CC as a consequence of increased secretion findings of different mitogenic responses of the two follic-
of hyaluronic acid and glycosaminoglycans that contribute ular GC subsets to growth factors and extend the findings
to the mucification of CC mass in response to these agents to include measurements of apoptosis in cells cultured un-
[8, 29]. Alterations in the composition of the ECM of COC der the same conditions. Disruption of cell-cell contact by
accompanying mucification could conceivably influence its cell dissociation decreases DNA synthesis and alters the
ability to bind IGFBPs, thus modifying the proliferative and response of CC to combined treatment with EGF and IGF-
antiapoptotic responses of CC to IGF-I. Recently reported I from mutual antagonism charactistic of intact COC to an
differences between MGC and COC in the pattern of additive response. Coculture of dissociated CC with DO
IGFBP secretion and retention in the ECM in response to increases DNA synthesis and response to each of the
mucifiying treatment offer support for this possibility [25]. growth factors alone in an additive manner but does not
Follicle-stimulating hormone by itself stimulated IGFBP3 restore their mutual antagonistic effects when added in
secretion by OCC but not MGC, an effect that was aug- combination. Unlike MGC, CC do not undergo apoptosis
mented by IGF-I. Both cell types also secreted IGFBP5, a if cultured as intact COC. Dissociation of both MGC and
greater proportion of which was retained in the ECM of COC triggers apoptosis in both cell subsets, and both IGF-
COC than of MGC, and combined treatment with FSH plus I and EGF are able to partially prevent the occurrence of
IGF-I increased the retention of IGFBP5 in the ECM of apoptosis in both cell subsets.
COC but not of MGC. [25].
In the present study, the previously reported differences ACKNOWLEDGMENTS
between CC and MCG in DNA synthesis induced by IGF-
I and EGF were abolished by cell dissociation. Differences We gratefully acknowledge the expert technical assistance of Ms. Les-
ley Ritter and Ms. Rebecca Thomas and the helpful suggestions of Dr.
in ECM composition in the two intrafollicular compart- Robert Gilchrist concerning oocyte-secreted factors.
ments may partially explain these functional differences.
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