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Regular Article |
a Istituto di Anatomia degli Animali Domestici, Università di Milano, Milan, Italy
b Reproductive Medicine Unit, 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 present experiments investigated the role of cell association in mediating 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 + 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 determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated 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 responses 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 characteristic 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 dramatic 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 cell-cell contact was a major factor reducing cell proliferation and inducing apoptosis among both subsets of GC.
apoptosis, cumulus cells, follicle, follicular development, granulosa cells, growth factors, ovary, pituitary hormones
INTRODUCTION
Cyclic ovarian follicular development is a complex process that involves proliferation, differentiation, and death of follicle cells [1]. Gonadotropins produced by the pituitary gland have a central role in the regulation of these processes. In addition, a wide range of paracrine and autocrine factors produced in the ovary have also been proposed as regulators of follicle growth.
During each estrous cycle, only one or a few follicles mature and ovulate in mammals while the majority of follicles 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, suggesting that GC die through an active process of programmed cell death or apoptosis [2, 3]. It has recently been suggested that cell-cell adhesion may affect the susceptibility of GC to apoptosis [4]. Moreover, recent studies demonstrated 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 important to appreciate the considerable heterogeneity in the somatic cell compartments of the follicle. As follicle development 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). Differences among GC subsets may be influenced both by proximity to blood supply (restricted to the thecal layer of the follicle) and by diffusion gradients of paracrine factors secreted into the follicular fluid by different follicle cell types including thecal cells, GC themselves, as well as the oocyte.
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 inhibitory 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 response 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 responded 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 attenuated 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 respective 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 32–36°C in PBS with antibiotic-antimycotic solution for up to 2 h during transport to the laboratory, pending follicular aspiration. Follicles 2–5 mm in diameter were aspirated with an 18-gauge needle on a 10-ml syringe containing a small volume of aspiration medium, M-199 containing 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 resuspended 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 suspension.
Cumulus cells and MGC were cultured either as intact COC and MGC aggregates or as dispersed cell preparations. Dispersion of both COC and mural cells was performed by the hypertonic method developed by Campbell [13], with slight modifications. Briefly, COC or GC aggregates were suspended in M-199 containing 0.2% BSA, 9.1 mM EGTA at pH 7.4, and incubated at 37°C 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 incubation. After dissociation, CC and MGC were washed twice in bicarbonate-buffered M-199C, counted with a hemocytometer, 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 x 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 (experiments 1 and 3) were carried out in 0.25 ml M-199C in 96-well flat-bottomed microwell tissue culture plates. Cumulus cells were cultured in wells containing either 8–10 intact COC estimated to contain 2.5–3.0 x 104 CC, or an equivalent number of dispersed CC, in 0.25 ml M-199C. Mural GC were cultured at 2.5 x 105 cells per well, either as cell aggregates or dissociated cells.
To evaluate the effects of oocyte-secreted factor(s) in 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 replicated 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 38.5°C in 5% CO2–95% air.
Assessment of DNA Synthesis
After 18 h of culture 0.1 µCi [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 manifold system (Millipore S.p.A., Milan, Italy), or with a Tomtec Cell Harvester 96 (Tomtec, Hamden, CT) onto glass fiber filters and then washed to remove soluble radioactivity. 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 Mannheim). The same concentration of cells used in DNA synthesis determination were plated before and after dissociation in an eight-chamber Lab-Tek slide (Nunc, Life Technologies, Milan, Italy) and cultured at 38.5°C in an atmosphere 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 37°C. Cells were fixed with 4% paraformaldehyde in PBS and stained using reagents 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 apoptotic cells in comparison to necrotic cells, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks. Cells were incubated 60 min at 37°C with the TUNEL mixture, allowing fluorescein-dUTP to label DNA strand breaks. The cells were then washed once with PBS and observed under epifluorescence and brightfield 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 experiment. The data were analyzed statistically by multivariate analysis of variance using SPSS, followed by Student-Newman-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 incorporation in CC and MGC are presented in Figure 1 for CC and Figure 2 for MGC.
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When added to cultures of intact COC or MGC aggregates, IGF-I resulted in a highly significant (P < 0.001) increase in [3H]thymidine incorporation, while only marginal, 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 divergence 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.
In the absence of growth factors (basal conditions), mean ± SEM [3H]thymidine incorporation in undissociated CC and MGC aggregates were 791 ± 135 and 104 ± 18 dpm/103 cells, respectively, compared to 120 ± 17 and 65 ± 6 dpm/103 cells, in dissociated CC and MGC. Cell dissociation 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 synthesis in both cell types in the presence of growth factors (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 responses 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 additive 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.
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Experiment 3: Effect of Growth Factors and Cell Dissociation on Apoptosis
Figure 3 shows immunohistochemical detection of apoptotic cells of aggregated and dispersed MGC.
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Results of apoptotic cell counts in intact aggregates and dispersed cell preparations of CC and MGC at time of isolation from follicles (0 h) and after 24 h of culture in different treatments are presented in Tables 2 and 3, respectively.
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At Time 0 h, between 1–2% of cells showed DNA fragmentation characteristic of apoptosis in both intact and dispersed CC and between 4–6% in both intact and dispersed MGC. A significant proportion of aggregated MGC (up to 23.0 ± 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 contact 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 gonadotrophic hormones [7, 14]. Growth factors seem to modulate the balance between GC survival and cell death. More than 99% of ovarian follicles undergo atresia during reproductive 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 apoptosis 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 possible involvement of the adhesion junction protein N-cadherin in triggering a signal transduction cascade that ultimately 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 [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 between different IGFBPs in sheep and bovine GC [24, 25]. Inhibitory effects of IGFBPs on both proliferative and secretory 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 enhance 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 augmented 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 compartments 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 components including bound IGFBPs associated with CC. The resulting absence of ECM-associated IGFBP5 may have altered 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 dissociation of the GC aggregates.
An additional possible contributing factor to the differences in responses of MGC and CC to growth factors and to cell dissociation may be oocyte-secreted factors. Considerable attention has focussed recently on regulatory actions of oocyte-secreted factors in a variety of differentiated responses of CC, including FSH- and EGF-induced cumulus expansion [29] and granulosa cell proliferation, based on studies involving removal of oocytes from COC (oocytectomy) and supplementation of granulosa cultures with oocyte-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 characteristic of MGC. Coculture of oocytectomized complexes with DO increased DNA synthesis and restored their relative 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 irrespective of the presence of both growth factors, whether alone or in combination. These findings indicate that the difference in response of oocytectomized versus dissociated CC complexes to IGF is attributable to loss of cell-to-cell contact and/or ECM components in the latter cell preparation, rather than to lack of oocyte-secreted factors. Further research is required to elucidate the complex interactions between oocyte-secreted factors, ECM with its associated IGF-binding proteins, and direct cell-to-cell contact in determining the differential phenotypes of different GC subsets 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 follicular GC subsets to growth factors and extend the findings to include measurements of apoptosis in cells cultured under 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.
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ACKNOWLEDGMENTS
We gratefully acknowledge the expert technical assistance of Ms. Lesley Ritter and Ms. Rebecca Thomas and the helpful suggestions of Dr. Robert Gilchrist concerning oocyte-secreted factors.
FOOTNOTES
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 University of Western Ontario, Canada. 
2 Correspondence: David T. Armstrong, Dept. of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville, S.A., Australia 5011. FAX: 61 8 8222 7521; david.armstrong{at}adelaide.edu.au
Accepted: July 24, 2000.
Received: January 27, 2000.
REFERENCES
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  T. S. Hussein, D. A. Froiland, F. Amato, J. G. Thompson, and R. B. Gilchrist Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins J. Cell Sci., November 15, 2005; 118(22): 5257 - 5268. [Abstract] [Full Text] [PDF]
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 Z. Zhao, D. Garbett, J. L Hill, and D. J Gross Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization Reproduction, October 1, 2005; 130(4): 517 - 528. [Abstract] [Full Text] [PDF]
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 Y. Pang and W. Ge Epidermal Growth Factor and TGF{alpha} Promote Zebrafish Oocyte Maturation in Vitro: Potential Role of the Ovarian Activin Regulatory System Endocrinology, January 1, 2002; 143(1): 47 - 54. [Abstract] [Full Text] [PDF]
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