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Regular Article |
a Department of Obstetrics and Gynecology, Fukui Medical University, Fukui 910-1193, Japan
b Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, follicular development, granulosa cells, ovary, theca cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have been studying the role of ovarian granulosa-theca cell interactions on folliculogenesis using a coculture system resembling, to some extent, the in vivo situation. Both types of cells are separated by an extracellular matrix in which epithelial (granulosa) and mesenchymal (theca) cells are grown on opposite sides of a collagen membrane (Fig. 1). In this in vitro model, the changes in these cells can be observed simultaneously. Also, cocultured cells possess morphological and functional characteristics that better resemble those of cells in the follicular wall in vivo than do theca and granulosa cells that are cultured separately [8]. We have demonstrated that the two follicular cell types reciprocally modulate their structure, function, and proliferation rate [8–10]. Our previous study showed that granulosa-theca cell interaction plays an important role in the control of follicular cell differentiation and that theca cells modulate the hormonal production capacity of granulosa cells in a biphasic manner during the ovarian follicular development process [11].
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Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. This process has been termed atresia, and during the past 8 yr, several research groups have reported that this massive destruction of follicular tissue has the biochemical and morphological characteristics of programmed cell death or apoptosis, which includes blebbing of the cell followed by DNA degradation (reviewed in [12, 13]). It ensures the selection of a dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple-embryo development during pregnancy and assures the development of few, but healthy, embryos [13]. It is suggested that theca cells may play an important role in controlling granulosa cell apoptosis [12]. In the present study, we have examined how theca cells control proliferation and apoptosis of granulosa cells isolated from different stages of follicular development using the in vitro coculture system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine ovaries were collected from heifers less than 15 min after slaughter at a local abattoir. Ovaries were placed in an ice-cold buffered salt solution and transferred to the laboratory less than 90 min after collection. The stage of the estrous cycle was determined morphologically, as previously described by Ireland et al. [14]. Follicular cells were prepared from the ovaries under sterile conditions, also as described previously [8]. Briefly, granulosa cells were harvested by aseptic needle aspiration from follicles and washed three times with a culture medium consisting of Waymouth MB 752/1 medium (Gibco, Grand Island, NY), Hanks solution (Gibco), and fetal calf serum (FCS; 6:3:1, v:v:v) (Gibco) supplemented with 100 µg/ml of streptomycin (Gibco) and 100 U/ml of penicillin (Gibco). Based on trypan blue-dye exclusion, cell viability was 30–35%.
For theca cell preparation, follicles with clear surfaces were cut into halves and the theca interna removed in situ with fine forceps. Granulosa cells, together with part of the theca cell layer, were removed by scraping with a scalpel under a stereomicroscope. The thin thecal layer thus obtained was minced and then treated with a Hanks-Hepes buffer containing collagenase (2150 U/ml, type 1; Sigma), and DNase (100 U/ml; Sigma), 0.4% (v/v) bovine serum albumin, and 0.2% (w/v) glucose (pH 7.4). Cell dissociation was allowed to continue for 30–60 min at 37°C with continuous stirring at 80 rpm and 0.25% (w/v) pancreatin (Sigma) in a Hanks-Hepes buffer for 7 min. Dispersed cells were washed three times; cell viability, as determined by the trypan blue-dye exclusion test, was 90–93%.
Culture of Granulosa and Theca Cells on Collagen Membrane
The membrane was made of type 1 collagen and had an area of 8 cm2. A supporting apparatus, to which a membrane (thickness, 
70 µm; Koken Co., Ltd., Tokyo, Japan) was attached, was placed in a 5-cm plastic dish. The apical and basal chambers were separated, and factors less than 12.5 kDa were permeable through a collagen membrane. To explore the effect of theca cells on granulosa cell functions, theca cells were plated onto membranes and cultured. Twenty-four hours later, each membrane was turned over, and granulosa cells were plated on the opposite side (cocultured). As controls, granulosa cells were cultured only on one side of a membrane (single-cultured).
Experimental Design
Small-follicle study In the first series of the present study, the effects of theca cells on apoptosis and proliferation of granulosa cells in the earlier stage of follicular development were investigated. For this study, ovaries with a regressing corpus luteum were used. Both granulosa and theca cells were collected from follicles (3–5 mm) that were supposed to be at a possible stage for recruitment into a follicular wave for further development [15, 16]. Healthy developing follicles were assessed according to the criteria previously established by Metcalf [17] for a vascularized pink theca externa and amber follicular fluid with no debris. In each study, the percentage of granulosa cells in sub-G1 phases of the cell cycle (apoptotic cells) was between 3 and 8%.
Theca cells (5 x 105 viable cells/dish) were plated onto a type 1 collagen membrane immediately after dispersal and cultured in a medium consisting of Waymouth MB 752/1 medium, Hanks solution, and FCS (6:3:1, v:v:v) supplemented with 100 U/ml of penicillin at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Twenty-four hours later, the collagen membrane was turned over, and granulosa cells (1 x 106 cells/dish) obtained from fresh ovaries were plated on the opposite side (cocultured). As controls, granulosa cells were cultured only on one side of the membrane (single-cultured). Following an additional 24-h culture in a medium containing 10% FCS, the cells were maintained in serum-free Ham F-12 medium supplemented with insulin (2 µg/ml) and transferrin (10 µg/ml), and media were replenished every 24 h.
After 24 h of serum-free culture, one-third of the dishes of single-cultured and cocultured groups were used for a study of cell-cycle analysis (for Day 1 of culture). Floating and attached granulosa cells were collected and combined to ensure complete representation of the cell population. Granulosa cells on the membranes were removed as follows: EDTA solution (0.2% EDTA in a 0.01 M phosphate buffer, pH 7.4) was added into the apical chamber in which granulosa cells were cultured. After 30 min of treatment, the apical surface of the membrane was washed three times with 1.5 ml of a 0.01 M phosphate buffer (pH 7.4). The detached cells were collected, centrifuged at 200 x g for 5 min, and resuspended in 2.0 ml of a 0.01 M phosphate buffer (pH 7.4). Cell numbers were calculated by means of a hemocytometer. The same study was also done at the end of Days 2 and 3 of the culture using the remaining single-cultured and cocultured dishes.
Large-follicle study In the second series of the present study, the effects of theca cells on apoptosis and cell proliferation of granulosa cells in the preovulatory stage were investigated. For this study, ovaries with a corpus luteum having a color of light yellow to white, a diameter of <1 cm, and an avascular surface were used. Both granulosa and theca cells were collected from follicles with clear fluid and a diameter of 15–18 mm [14–16, 18]. Theca cells (5 x 105 viable cells/dish) and granulosa cells (1 x 106 cells/dish) were plated on the membrane, and the study was performed as described for the small-follicle study.
Crossover study In the third series of the present study, we investigated how theca cells from large follicles modulate apoptosis and cell proliferation of granulosa cells from small follicles. The effects of theca cells from small follicles on apoptosis and cell proliferation of granulosa cells from large follicles were also examined. First, granulosa cells from small follicles were cultured with theca cells from large follicles. Second, granulosa cells from large follicles were cultured with theca cells from small follicles. In both studies, granulosa cells were cultured only on one side of the membrane as controls.
Cell-Cycle Analysis by Flow Cytometry
Cell-cycle analysis of granulosa cells was performed as described previously [19]. Briefly, granulosa cells collected from media and membrane surface were centrifuged and fixed in cold methanol (-20°C) for 1 h. Subsequently, cells were centrifuged at 300 x g for 5 min, resuspended in 0.5 ml of cold PBS at 4°C, and stained for at least 15 min with 50 µg/ml of propidium iodide in the presence of RNase A (100 µg/ml). Cells were then analyzed in the fluorescence-activated cell sorter (Epics XL; Beckman Coulter Inc., Fullerton, CA). Ten thousand events from the gated subpopulation were recorded separately. A percentage of granulosa cells in each sub-G1 (apoptotic cells), G0/G1, S, and G2/M (cells in proliferation) phases of the cell cycle was calculated from the DNA histogram data using Coulter software.
Statistical Analysis
Each study was repeated three times on different days. Data are presented as the mean ± SD of the three experiments, each with three replicate culture dishes on each day of the culture. Data on cell number and a percentage of granulosa cells in each apoptotic and proliferative phases were analyzed from Day 1 to Day 3 of the culture to evaluate dynamic changes of these responses in single-cultured and cocultured groups. Two-factor factorial ANOVA was performed. When a significant effect was detected (P < 0.05), Tukey-Kramer test for intergroup comparison was performed. All statistical analysis was performed using a statistical analysis program (StatView version 5.0; SAS Institute, Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Granulosa cell numbers on Days 1–3 of the serum-free culture are shown in Figure 2. In the small-follicle study, the numbers of single-cultured cells on Days 2 and 3 were smaller than that on Day 1 of culture (P < 0.05). Such a difference was not detected in the cell numbers of the cocultured group. When granulosa cell numbers of single-cultured and cocultured groups were compared on Day 3, that of the cocultured group was significantly larger than that of the single-cultured group (5.8 ± 0.4 vs. 3.7 ± 0.7 x 105 cells, P < 0.05) (Fig. 2A).
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In the large-follicle study, the granulosa cell numbers on Days 2 and 3 of culture were smaller than those on Day 1 in both single-cultured and cocultured groups (P < 0.05). No significant difference was detected in cell numbers between single-cultured and cocultured groups throughout the culture period (Fig. 2B).
In the crossover study, the numbers of granulosa cells from small follicles were significantly increased in the presence of theca cells from large follicles on Day 3 of the culture (0.6 ± 0.5 vs. 3.1 ± 0.3 x 105 cells, P < 0.05) (Fig. 2C). In contrast, the number of granulosa cells from large follicles was not affected by the presence of theca cells from small follicles (Fig. 2D).
Apoptosis
The percentage of granulosa cells in sub-G1 phase (apoptotic cells) during the course of culture is shown in Figure 3, A and B. In the small-follicle study, when the granulosa cells were cultured alone, the percentage of apoptotic cells on Day 3 of culture was higher than those on Days 1 and 2. In contrast, when the granulosa cells were cultured with theca cells, the percentage of apoptotic cells on Day 3 of culture was lower than that on Day 1. Moreover, the percentage of apoptotic cells in the cocultured group was reduced by 1.8-fold compared to that of the single-cultured group on Day 3 of culture (3.4% ± 1.0% vs. 13.3% ± 2.5%, P < 0.05) (Fig. 3A).
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In the large-follicle study, the percentage of apoptotic granulosa cells declined on Days 2 and 3 of culture in both single-cultured and cocultured groups (Fig. 3B). The percentage of apoptotic cells in the cocultured groups was lower than that of the single-cultured groups on Day 1 of culture (7.2% ± 1.0% vs. 11.3% ± 1.1%, P < 0.05). However, no significant difference was detected between single- and cocultured groups on Days 2 and 3 of culture.
In the crossover study, the percentage of cells in sub-G1 phase of granulosa cells from small-sized follicles was significantly reduced in the presence of theca cells from large-sized follicles on Day 3 of culture (15.8% ± 2.1% vs. 10.9% ± 0.8%, P < 0.05) (Fig. 3C). No such difference was detected between single-cultured and cocultured groups when granulosa cells from large-sized follicles were cultured with theca cells from small-sized follicles (Fig. 3D).
Cell Proliferation
The percentages of granulosa cells in S and G2/M (cells in proliferation) phases during the course of culture are shown in Figure 4. The percentage of cells in the proliferative phase was unchanged throughout the culture period in small- and large-follicle studies and crossover studies. No significant difference was detected between single-cultured and cocultured groups in all the studies.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study demonstrated that theca cells protect granulosa cells from apoptosis and that the effect of theca cells on apoptosis of granulosa cells changes during follicular maturation. In the small follicle, apoptosis in granulosa cells, which gradually increased with time of culture, was totally suppressed by the presence of theca cells. This suggests that theca cells may secrete a survival factor which overcomes apoptotic death of granulosa cells. In the large follicle, apoptosis of granulosa cells was suppressed by the presence of theca cells on Day 1 of culture; however, such an effect of theca cells was not detected on Days 2 and 3 of the culture. We suggest that granulosa cells from large follicles either may produce survival factors or may no longer produce factors that may cause apoptosis. It seems that the thecal factors are not essential for the survival of granulosa cells at this stage of follicular development. These data would support the notion that the potential of granulosa cells to undergo apoptosis is changed during follicular maturation.
Yang and Rajamahendran [29] reported that the rate of DNA fragmentation in the culture of bovine granulosa cells from small follicles was higher than that of such cells from medium and large follicles. Moreover, they showed an elevated ratio of Bax to Bcl-2 expression in the atretic follicle [30]. A similar pattern of Bcl-2 and Bax expression has been reported in rat follicles at the mRNA level [31].
In each estrous cycle in the ovary of mono-ovulatory species, a cohort of follicles is recruited to begin the final steps of growth and maturation to the preovulatory stage of development. However, only one follicle will be selected as the dominant follicle for continued survival and, ultimately, ovulation, whereas the remaining subordinate follicles in the cohort undergo the degenerative process of atresia, resulting from an induction of apoptosis in the granulosa cells. The small follicles used in this experiment were expected to be recruited into the cohort of maturing follicle, indicating that the natural fate of most of these granulosa cells is entering into the apoptotic process. Thus, it is possible that the interaction between granulosa and theca cells is one of the important mechanism(s) by which one follicle is selected as a leading one. Disconnection of the communication between granulosa and theca cells by some unknown mechanism(s) might occur in follicles that enter the atretic process. It is also possible that granulosa cells acquire survival potential after selection of a leading follicle.
Two explanations are possible for the results showing the different effects of theca cells on apoptosis of granulosa cells in the small- and large-follicle studies. One possibility is that theca cells of small and large follicles produce different factor(s) affecting apoptosis of granulosa cells. The other possibility is that theca cells send the same factor(s) to granulosa cells throughout the follicular maturation process and, in turn, that granulosa cells from small and large follicles show different types of responses to the same thecal factor(s). A crossover study was performed to answer this question. Apoptosis of granulosa cells from small follicles was also suppressed by the presence of theca cells from large follicles in the same manner as in the small-follicle study. Apoptosis of granulosa cells from large follicles was suppressed by the presence of theca cells from small follicles in the same manner as in the large-follicle study. Thus, we speculate that theca cells send the same factor(s) to granulosa cells throughout the follicular maturation process and, in turn, that granulosa cells respond differently to this as they differentiate in the process of follicular maturation. This hypothesis was also supported by the results of our previous study, in which the role of theca cells in control of the hormone-producing activity of granulosa cells was investigated. Hormone production of immature granulosa cells was reduced by the presence of theca cells from either small or large follicles, whereas that of granulosa cells from matured follicles was augmented by theca cells irrespective of their origin [11].
Recent studies also suggest the importance of granulosa and theca cell interactions in the regulation of apoptosis of granulosa cells. Epidermal growth factor, transforming growth factor 
, and keratinocyte growth factor (KGF) appear to be potential physiological inhibitors of apoptotic cell death in the rat ovary [12, 20, 32]. The existence of a positive-feedback loop that is mediated by kit ligand/stem cell factor (KL) and hepatocyte growth factor (HGF), and HGF was also identified between theca and granulosa cells in the bovine [7]. These growth factors produced by theca cells might be one of the factors that decreased the incidence of apoptosis in granulosa cells during the present study.
Granulosa cell numbers of the cocultured group were significantly larger than those of the single-cultured group in the small-follicle study. However, no significant difference was detected in the percentage of granulosa cells in S and G2/M phases between single-cultured and cocultured groups. Thus, the difference in the rate of apoptotic cells between single-cultured and cocultured groups reflects the difference in cell numbers between single-cultured and cocultured groups. Meanwhile, in the large-follicle study, a significant difference was detected neither in cellular proliferation nor in rate of apoptosis between single-cultured and cocultured groups; thus, no significant difference was detected in cell numbers between single-cultured and cocultured groups.
In the present study, we examined how theca cells control apoptosis and proliferation of granulosa cells in the process of follicular development using an in vitro coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. The study showed that theca cells have an important role in regulating apoptosis of granulosa cells. The paracrine factors secreted from theca cells may inhibit apoptosis of granulosa cells. Moreover, the cellular susceptibility to apoptosis is different between early and late-stage granulosa cells. These findings support our previous hypothesis that cross-talk between granulosa and theca cells is essential for maintenance of the physiological function of follicular cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants-in-Aid 05557072, 05857172, 06454467, and 10671527 from the Ministry of Education, Science, and Culture of Japan. 
2 Correspondence: Fumikazu Kotsuji, Department of Obstetrics and Gynecology, Fukui Medical University, Matsuoka-Cho, Yoshida-Gun, Fukui 910-1193, Japan. FAX: 81 776 61 8117; kotsujif{at}fmsrsa.fukui-med.ac.jp
Accepted: December 27, 2001.
Received: September 12, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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