| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Department of Obstetrics and Gynecology, Fukui Medical University, Matsuoka-Cho, Yoshida-Gun,Fukui 910-1193, Japan
b Department of Laboratory Animal Science, School of Veterinary Medicine and Animal Sciences, Kitazato University, Towada, Aomori 034, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We have successfully developed a coculture system in which epithelial and mesenchymal cells are grown on opposite sides of a collagen membrane (Fig. 1). The advantages of this in vitro model are that 1) it resembles the in vivo environment, in which the two types of cells are separated by an extracellular matrix, and 2) the changes in these cells can be observed simultaneously. In addition, this system has made it possible to study the possible role of intercellular communication in follicular cell morphology and function [9]. Using this coculture system, we previously demonstrated that the two follicular cell types reciprocally modulate their proliferation, morphology, structure, and function and that cocultured cells, in comparison to cells cultured alone, possess morphological and functional characteristics that better resemble those of cells in the follicular wall in vivo. Moreover, our observations also suggest that granulosa-theca cell interactions are essential for the maintenance of normal follicular structure and function [9–11].
|
In the present study, we have examined how theca and granulosa cell interaction controls the hormone-producing activity and growth of granulosa and theca cells in the process of follicular development using an in vitro coculture system.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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 60 min after collection. Follicular cells were prepared from bovine ovaries as described previously [9]. Briefly, granulosa cells were harvested by aseptic needle aspiration from follicles and washed three times with a culture medium consisting of Waymouth's MB 752/1 medium (Gibco, Grand Island, NY), Hanks' solution (Gibco), and 10% fetal calf serum (FCS) (6:3:1, v:v:v) supplemented with 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco). On the basis of 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 layer was then removed with fine forceps. Granulosa cells, together with a part of the theca cell layer, were removed by scraping with a scalpel under a stereomicroscope. The thin theca layer thus obtained was minced and then treated with a Hanks’-Hepes buffer containing collagenase (2150 U/ml, type 1; Sigma Chemical Co., St. Louis, MO) and DNase (100 U/ml; Sigma), 0.4% BSA, and 0.2% glucose (pH 7.4). Cell dissociation was allowed to continue for 30–60 min at 37°C with continuous stirring at 80 rpm and with 0.25% 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%.
Experimental Design: Small-Follicle Study
In the first series of the study, the effects of theca and granulosa cell interaction on the hormone-producing activity and growth of granulosa and theca cells in the earlier stage of follicular development were investigated. For this study, ovaries without a corpus luteum were used. Both granulosa and theca cells were collected from follicles 3–5 mm in diameter that were supposed to be at a possible stage for recruitment into a follicular wave for further development [12, 13].
Effect of theca cells on granulosa cell function and growth For exploration of the effect of theca cells on granulosa cell functions, theca cells (5 x 105 viable cells per dish) were plated onto a type 1 collagen membrane immediately after dispersal and cultured in a culture medium consisting of Waymouth's MB 752/1 medium (Gibco), Hanks' solution (Gibco), and 10% FCS (6:3:1, v:v:v), supplemented with 100 U/ml 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 freshly prepared granulosa cells (1 x 106 cells per dish) were plated on the opposite side (cocultured). As controls, granulosa cells were cultured only on one side of the membrane (single-cultured). After an additional 24-h culture in a medium containing 10% FCS, the cells were maintained in serum-free Ham's F-12 supplemented with insulin (2 µg/ml), transferrin (10 µg/ml), and testosterone (10-8 mol/L).
After 48 h of serum-free culture, half of the dishes of single-cultured and cocultured groups were used for a study of hormonal production and granulosa cell growth (for the first 48 h of culture). Culture medium was collected from the apical and basal chambers and stored at -20°C for hormonal measurement. Granulosa cells were collected from the membranes.
The culture medium of the remaining dishes was replenished, and the serum-free culture was continued for another 48-h period. The dishes were then analyzed for hormonal production and granulosa cell growth for the second 48 h of culture.
Effect of granulosa cells on theca cell functions For exploration of the effect of granulosa cells on theca cell functions, granulosa cells were plated onto a collagen membrane on the first day of culture. Freshly prepared theca cells were plated on the opposite side 24 h later (cocultured). As controls, theca cells were cultured only on one side of the membrane (single-cultured). The culture was carried out in the same way as described above. Half of the dishes of both single-cultured and cocultured groups were used for the analysis of hormonal production and theca cell growth for the first 48 h of serum-free culture. The remaining dishes of each group were analyzed for hormonal production and theca cell growth for the second 48 h of culture.
Experimental Design: Large-Follicle Study
In the second series of the study, the effects of cellular interaction on the hormone production and proliferation activities of granulosa and theca cells in the preovulatory stage were investigated. For this study, ovaries with a corpus luteum light yellow to white in color, < 1 cm in diameter, and avascular surface were used. Both granulosa and theca cells were collected from follicles with clear fluid and a diameter of 15–18 mm [12–15]. The study was performed as described for the small-follicle study.
Experimental Design: Crossover Study
In the third series of the study, we investigated how follicular cells from large follicles modulate the hormonal production and growth of follicular cells from small follicles. The effects of follicular cells from small follicles on the hormonal production and growth of follicular cells from large follicles were also examined. Firstly, granulosa cells from small follicles were cultured with theca cells from large follicles, and theca cells from small follicles were cultured with granulosa cells from large follicles. Secondly, granulosa cells from large follicles were cultured with theca cells from small follicles, and theca cells from large follicles were cultured with granulosa cells from small follicles. In both studies, granulosa or theca cells were cultured only on one side of the membrane as controls.
Measurement of Hormone Levels in Culture Media
Estradiol, progesterone, inhibin, and androstenedione concentrations in the serum-free media of the first and second 48-h cultures were measured by a double-antibody RIA. For the estradiol, progesterone, and inhibin assay, media collected from the granulosa cell side were used. For androstenedione assay, the medium from the theca cell side was used. All samples were analyzed in the same assays, and the intraassay coefficients of variation for estradiol, progesterone, inhibin, and androstenedione were 7.3%, 6.4%, 5.8%, and 6.0%, respectively [9, 16]. The data were normalized by cell number.
Calculation of Granulosa and Theca Cell Number
Granulosa and theca cells on these membranes were removed by trypsin treatment (1% trypsin and 0.2% EDTA in a 0.01 M phosphate buffer, pH 7.4) and analyzed for cell growth as described previously [9]. Briefly, trypsin-EDTA solution was added into the apical chamber in which either granulosa or theca cells were being cultured. After the 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, and resuspended in 2.0 ml of a 0.01 M phosphate buffer (pH 7.4). The viability of both granulosa and theca cells after removal from the collagen membrane was 96–98%. Cell numbers were calculated by means of a hemacytometer.
Statistical Analysis
Each study was repeated three times on different days. Data are presented as the mean ± SEM of the three experiments, each with three replicate culture dishes. Results were analyzed by a two-way ANOVA, and the difference between experimental groups was tested by Tukey's test. In the crossover study, the Student's t-test was used. A P value of less than 0.05 was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Granulosa and theca cell numbers after the first and second 48 h of culture are summarized in Figure 2.
|
Granulosa cell growth In both small- and large-follicle studies, there was no significant difference in granulosa cell numbers between single-cultured and cocultured groups (Fig. 2A). In the crossover study as well, no significant difference was detected in the numbers of granulosa cells between single-cultured and cocultured groups (data not shown).
Theca cell growth The theca cell numbers in cocultured groups were significantly larger than those in single-cultured groups in both small- and large-follicle studies (Fig. 2B). In the crossover study, the theca cell numbers in cocultured groups were significantly larger than those in single-cultured groups as well (data not shown).
Estradiol, Progesterone, and Inhibin Production by Granulosa Cells
The production of estradiol, progesterone, and inhibin per 1 x 105 granulosa cells during the first and second 48 h of serum-free culture is summarized in Figure 3 and Table 1. Our preliminary study showed that single-cultured theca cells from both small and large follicles produced negligible amounts of progesterone (the progesterone-producing ability of theca cells from small and large follicles was less than 5% and 6%, respectively, of that of single-cultured granulosa cells) and did not produce estradiol or inhibin. This suggests that granulosa cells are the primary source of these hormones under this experimental condition. In coculture dishes, inhibin was detected in the granulosa cell- but not in the theca cell-conditioned medium, and no difference was detected in estradiol, progesterone, or androstenedione concentrations between the media in the apical and basal chambers.
|
|
In the small-follicle study, no significant difference was detected in estradiol, progesterone, or inhibin production between single-cultured cells and cocultured cells during the first 48 h of serum-free culture. During the second 48 h of culture, however, the production of these hormones by granulosa cells cultured with theca cells was significantly lower than that by single-cultured cells (estradiol, 14 ± 5%; progesterone, 64 ± 6%; inhibin, 27 ± 4% of production by single-cultured cells, respectively). During the second 48 h of culture, hormonal production by single-cultured cells increased to 3.4-fold (estradiol), 1.6-fold (progesterone), and 2.9-fold (inhibin) that observed during the first 48 h of culture, respectively. By contrast, no significant difference was detected in hormonal production by the cocultured cells between the first and the second 48 h of culture (Fig. 3A).
In the large-follicle study, no significant difference was detected in the production of these hormones between single-cultured and cocultured cells during the first 48 h of culture. However, hormonal production by granulosa cells cultured with theca cells was significantly higher than that by single-cultured cells during the second 48 h of culture (estradiol, 253 ± 34%; progesterone, 156 ± 24%; inhibin, 287 ± 45% of production by single-cultured cells, respectively). During the second 48 h of culture, hormonal production by single-cultured cells was reduced to 51 ± 12% (estradiol), 62 ± 9% (progesterone), and 35 ± 12% (inhibin) of that during the first 48 h, respectively. By contrast, no significant difference was detected in the hormonal production by the cocultured cells between the first and the second 48 h of culture (Fig. 3B).
In the crossover study also, no significant difference was detected in the production of these hormones between single-cultured and cocultured cells during the first 48 h of culture, irrespective of the origin of the follicular cells. During the second 48 h of culture, hormonal production by granulosa cells from small follicles was significantly reduced by the presence of theca cells from large follicles. Hormonal production by the granulosa cells from large follicles was significantly augmented by the presence of theca cells from small follicles (Table 1).
Androstenedione Production by Theca Cells
Single-cultured granulosa cells did not produce androstenedione under this experimental condition, suggesting that theca cells are the primary source of this hormone. No difference was detected in androstenedione concentrations between the media in the apical and basal chambers of the cocultured groups.
Figure 4 shows the production of androstenedione per 1 x 105 theca cells during the first and second 48 h of serum-free culture. In both small- and large-follicle studies, the production of androstenedione by the theca cells of the cocultured groups was significantly higher than that by single-cultured cells (small-follicle study, first 48 h: 682 ± 266%; second 48 h: 861 ± 190%; large-follicle study, first 48 h: 467 ± 143%; second 48 h: 1298 ± 414%).
|
In the small-follicle study, no significant difference was detected in androstenedione production between the first and the second 48 h of culture in either single-cultured or cocultured groups. In the large-follicle study, androstenedione production by cocultured cells during the second 48 h of culture was 3.1-fold higher than that during the first 48 h of culture. However, no significant difference was detected in the androstenedione production by the single-cultured cells between the first and the second 48 h of culture (Fig. 4).
In the crossover study, the production of androstenedione by theca cells was augmented by the presence of granulosa cells as well, irrespective of the origin of the cells (Table 1).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
It is generally accepted that follicular cells luteinize when they are cultured in vitro. In the present study, granulosa cells from small-sized follicles produced progesterone, indicating that the luteinization process of these cells proceeded to a certain extent during the culture. Thus, this in vitro experiment may not exactly mimic the phenomena in vivo since granulosa cells produce negligible amounts of progesterone in the early stage of follicular maturation in vivo. In spite of this limitation of the in vitro experimental system, it is possible to conclude that the theca factor(s) inhibits the differentiation of granulosa cells at the early stage of follicular development for the reasons described below. Hormonal production by single-cultured cells from small-sized follicles was dramatically augmented during the second 48 h of culture, indicating that the luteinization (differentiation) process of single-cultured granulosa cells proceeded further during this period. By contrast, no such augmentation was observed when granulosa cells were cultured with theca cells, indicating that the differentiation of granulosa cells was inhibited by cocultured theca cells. From these observations, we speculate that the theca factor(s) is crucial in restraining the differentiation of granulosa cells in the early stage of follicular maturation in vivo and that elimination of the effects of theca cells produces the luteinization of cells when they are cultured in vitro.
We previously demonstrated that the proliferation of immature granulosa cells cultured in a serum-containing medium was significantly enhanced by the presence of theca cells [9]. This effect of the theca cells, however, was not observed in the present study. While the reasons for this apparent discrepancy are unknown, a possible explanation is that the presence of serum in the culture is essential for expression of the growth-promoting effect of theca cells. Supporting this possibility is the observation that epidermal growth factor, which is closely related to transforming growth factor-
produced by bovine theca cells [17] and which shares the same cell-membrane receptor [18], is increasingly mitogenic in the presence of elevated serum concentrations in bovine granulosa cells [19]. Maximum cell division was achieved by the epidermal growth factor acting synergistically with other growth factors found in serum [20]. Likewise, the theca cell factor(s) stimulated granulosa cell growth in a serum-containing medium [21, 22]. In contrast, when granulosa cells from large-sized follicles were cultured with theca cells, an enhancement of the proliferation of granulosa cells by theca cells was not detected even in the presence of serum (data not shown). It is well recognized that the proliferative rate of granulosa cells increases markedly when the theca layer comes outside of the basement membrane and blood vessels appear in the theca layer [8]. Considering all these results, we propose the hypothesis that the appearance of theca cell layers and the blood vessels in them are key factors initiating the proliferation of granulosa cells in the early stage of follicular development. Moreover, we suggest that granulosa cells lose the ability to respond to the growth-promoting activity of theca factor(s) when they are fully differentiated in the late stage of follicular development.
There are two possible explanations for the results showing the biphasic effects of theca cells on the hormonal production capacity of granulosa cells. One possibility is that theca cells of small- and large-sized follicles produce different factor(s) affecting the hormonal production of granulosa cells. The other possibility is that theca cells send the same factor(s) to granulosa cells throughout the follicular maturation process and that granulosa cells from small- and large-sized follicles show different types of responses to the same theca factor(s). A crossover study was performed to address this issue. Hormonal production by granulosa cells from small-sized follicles was also suppressed by the presence of theca cells from large-sized follicles, and that by granulosa cells from large-sized follicles was augmented by the presence of theca cells from small-sized follicles. Thus, we speculate that theca cells send the same factor(s) to granulosa cells throughout the follicular maturation process and that granulosa cells respond differently to this as they differentiate in the process of follicular maturation.
It is of interest that there was no difference in the hormonal production by granulosa cells between single-cultured and cocultured groups during the first 48 h of culture but that the difference became significant during the second 48 h of culture. It is possible that the in vivo influence of the theca cells remained during the first 48 h of culture even in the single-cultured cells and that the difference in hormonal production between the single- and the cocultured cells was small. In the small-follicle study, removal of the influence of the theca cells in vivo resulted in an increase in hormonal production by the single-cultured cells during the second 48 h of culture, which was suppressed by the theca cells in the cocultured system. Likewise, in the large-follicle study, removal of the influence of the theca cells in vivo resulted in a decrease in hormonal production during the second 48 h of culture, as observed in the single-cultured cells. The decrease was attenuated by the presence of theca cells in the cocultured system.
Identification of the factor(s) involved in the theca-granulosa cell communication in the control of granulosa cell differentiation remains to be achieved. Numerous follicular factors such as steroids, growth factors, cytokines, and extracellular matrix molecules have been shown to modulate basal and gonadotropin-induced hormonal production by follicular cells in a paracrine fashion, and the responsiveness of the granulosa cells to these factors appears to differ with respect to species, developmental stage, and culture conditions [1–7, 21–25]. Several studies support the hypothesis that theca cells control the functions and growth of granulosa cells in a manner dependent on the stage of follicular development [10]. For instance, Voss and Fortune reported that the effect of coculture on oxytocin production by bovine granulosa and theca cells was more than additive when granulosa cells were isolated early in the follicular phase, but the effect of the coculture was additive only when granulosa cells were obtained in the mid-follicular phase [26]. Wang et al. [27] reported that granulosa and theca cells interact to regulate the chicken follicular plasminogen activator system at the late but not the early developmental stage of follicles. Peddie et al. [28] reported that epidermal growth factor/transforming growth factor- produced by theca cells augments the proliferation and suppresses the differentiation of granulosa cells in the early stage of follicular maturation, while it augments the differentiation of the granulosa cells in the late stage. Parrott and Skinner [6] showed that theca cells produce a keratinocyte growth factor (KGF) regulating granulosa cell growth and functions and that expressions of the KGF in theca cells and of the KGF receptor in granulosa cells are higher in large follicles than in small or medium-sized follicles. It is unlikely that the action of a single factor can account for all the findings of the present study. We speculate that a number of intragonadal regulators interact with one another and control the functional differentiation of granulosa cells in a complex manner during follicular maturation. The mechanism(s) producing the change in the granulosa cell response to the theca factor(s) during follicular development process remains to be investigated.
In summary, we have examined the influence of granulosa-theca cell interaction on the hormonal production and proliferation activities of follicular cells from early- and late-developmental-stage follicles using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. The study showed that granulosa-theca cell interaction is a major follicular constituent in the control of follicular cell differentiation and proliferation during ovarian follicular development. The theca factor(s) inhibits the differentiation of granulosa cells at the early stage of follicular development but promotes it during late follicular maturation, while the granulosa factor(s) promotes both differentiation and growth of theca cells throughout the follicular maturation process. Our present and previous [9] studies also suggest that the theca factor(s) promotes granulosa cell growth with the aid of serum in the early stage of follicular development; however, granulosa cells lose their responsiveness to the growth-promoting activity of theca factor(s) in the late stage of follicular development. These findings support our previous hypothesis that cross-talk between granulosa and theca cells is essential for the maintenance of the physiologic function and structure of follicular cells.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 81 776 61 8117; kotsujif{at}fmsrsa.fukui-med.ac.jp
Accepted: August 9, 1999.
Received: October 30, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
by theca cells. Endocrinology 1988; 123:2632–2638.[Abstract]This article has been cited by other articles:
|
|
 |
|
  A Bonnet, R Dalbies-Tran, and M A Sirard Opportunities and challenges in applying genomics to the study of oogenesis and folliculogenesis in farm animals Reproduction, February 1, 2008; 135(2): 119 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tajima, M. Orisaka, K. Hosokawa, A. Amsterdam, and F. Kotsuji Effects of Ovarian Theca Cells on Apoptosis and Proliferation of Granulosa Cells: Changes During Bovine Follicular Maturation Biol Reprod, June 1, 2002; 66(6): 1635 - 1639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. De Geyter, M. De Geyter, P. R. Huber, E. Nieschlag, and W. Holzgreve Progesterone serum levels during the follicular phase of the menstrual cycle originate from the crosstalk between the ovaries and the adrenal cortex Hum. Reprod., April 1, 2002; 17(4): 933 - 939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Mizutani, K. Yamada, T. Yazawa, T. Okada, T. Minegishi, and K. Miyamoto Cloning and Characterization of Gonadotropin-Inducible Ovarian Transcription Factors (GIOT1 and -2) That Are Novel Members of the (Cys)2-(His)2-Type Zinc Finger Protein Family Mol. Endocrinol., October 1, 2001; 15(10): 1693 - 1705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. Slee, S. G. Hillier, P. Largue, C. R. Harlow, G. Miele, and M. Clinton Differentiation-Dependent Expression of Connective Tissue Growth Factor and Lysyl Oxidase Messenger Ribonucleic Acids in Rat Granulosa Cells Endocrinology, March 1, 2001; 142(3): 1082 - 1089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. A. Dissen, J. A. Parrott, M. K. Skinner, D. F. Hill, M. E. Costa, and S. R. Ojeda Direct Effects of Nerve Growth Factor on Thecal Cells from Antral Ovarian Follicles Endocrinology, December 1, 2000; 141(12): 4736 - 4750. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
