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a Department of Obstetrics and Gynecology, and
b Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520-8063
c Department of Gynecology and Obstetrics, Karol Marcinkowski University School of Medical Science, Poznan, Poland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence point to insulin-like growth factor-I (IGF-I) and platelet-derived growth factor (PDGF) as potentially important regulators of proliferation and differentiation of T-I cells. First, IGF-I and PDGF play a role in the control of developmental growth, homeostatic maintenance, and differentiation of a broad range of other tissues [6, 7]. Second, T-I cells possess receptors for IGF-I and PDGF, and hence, actions of these growth factors are biologically feasible [8–11]. Third, IGF-I modulates many aspects of T-I cell functions. In particular, IGF-I stimulates cellular differentiation: it promotes androgen production by increasing the expression of messenger ribonucleic acid for cholesterol side-chain cleavage, 3ß-hydroxysteroid dehydrogenase, and 17
-hydroxylase [12–14]. Furthermore, our recent studies have demonstrated that IGF-I stimulates proliferation of T-I cells in rats and humans [15, 16].
Proliferative actions of IGF-I may be modulated by PDGF. The traditional model of mechanisms regulating mitotic activity of mesenchymal cells describes dual control of proliferation by "competence" and "progression" factors [17, 18]. According to this model, IGF-I acts as a progression factor inducing competent cells to synthesize DNA. PDGF, on the other hand, has been described as a competence factor capable of stimulating the cells to progress from G0 to G1 phase of the cell cycle, but not beyond. PDGF may also affect other aspects of cellular function, including modulation of differentiation [6].
Proliferation and differentiation are thought to occur sequentially, with division of undifferentiated cells followed by differentiation and loss of proliferative activity [19]. In accord with this paradigm, proliferation and terminal differentiation are mutually exclusive; furthermore, organogenesis and folliculogenesis may be viewed as processes whereby a progressive decrease of proliferative activity occurs in parallel with the development of terminal differentiation. For T-I cells, steroidogenic activity is considered a measure of differentiation [13, 14]. Little, however, is known about the role of differentiation in the modulation of proliferative activity and about mechanisms regulating the development of steroidogenically active and inactive subpopulations of T-I cells.
The present study was designed to determine whether steroidogenic activity affects proliferation of T-I cells and to evaluate the effects of PDGF in comparison to IGF-I on the proliferation of steroidogenically active and steroidogenically inactive cells. To our knowledge, this is the first report demonstrating that differentiated (steroidogenically active) T-I cells proliferate and that individual growth factors selectively affect proliferation of different subpopulations of these cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following materials were purchased from Sigma Chemical Co. (St. Louis, MO): Medium-199 with Hanks' Balanced Salt Solution (HBSS), medium 199 with HBSS (10x), McCoy's 5a medium (modified, without serum), L-glutamine, BSA, trypsin-EDTA (0.05%/0.02%), nitro-blue tetrazolium, 5ß-androstan-3ß-ol-17-one, ß-NAD+, sesame oil, Percoll, recombinant IGF-I, Hoechst 33342, and DiOC5. Collagenase type I (Clostridium histolyticum, CLS1; 146 U/mg) and DNase I (bovine pancreas; 2298 U/mg) were obtained from Worthington Biochemical Co. (Freehold, NY). The following materials were purchased from Grand Island Biological Co. (Grand Island, NY): Trypan blue dye (0.4%, w:v), antibiotic-antimycotic preparation (penicillin, 10 000 IU/ml; streptomycin, 10 000 µg/ml; amphotericin B, 25 µg/ml), and Dulbecco's PBS (1x, pH 7.2, without MgCl2 and CaCl2). HEPES was purchased from American Bioanalytical (Natick, MA). Radiolabeled [3H]thymidine was purchased from Amersham Life Sciences Inc. (Arlington Heights, IL). Recombinant PDGF-AB heterodimer and anti-human PDGF neutralizing antibody (Ab-PDGF) were purchased from R&D Systems (Minneapolis, MN).
Animals
Immature (25 days old) female Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY) and housed with a 12L:12D photoperiod in an air-conditioned environment. Standard rat chow and water were given ad libitum. Starting on the 28th day of age, the animals received injections of estradiol-17ß (1 mg/0.3 ml sesame oil s.c.) daily for three days in order to stimulate ovarian development. On the morning after the last injection (Day 31 of age) the animals were anesthetized with ketamine and xylazine (i.p.) and killed by perfusion with 0.9% saline. All treatments and procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and a protocol approved by the Yale University Animal Care Committee, and the investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Isolation of Theca-Interstitial Cells and Cell Cultures
Ovaries were dissected, and T-I cells were isolated and purified as described previously [15]. The cells were counted using a hemocytometer, and viability was determined using a trypan blue dye exclusion test. Cell viability was in the 85–95% range. The cells were cultured in McCoy's 5a medium supplemented with L-glutamine (2 mM), BSA (1 mg/ml), penicillin (10 000 IU/ml), streptomycin (10 000 µg/ml), and amphotericin B (25 µg/ml). Cultures were carried out at 37ÅC in an atmosphere of 5% CO2 in humidified air in 6-well plates, 24-well plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ), or 96-well plates (Corning Glass Works, Corning, NY) for 48 h. The final amount of T-I cells plated was 700 x 103 cells/2 ml in 6-well plates, 350 x 103 cells/ml in 24-well plates, and 35 x 103 cells/0.25 ml in 96-well plates. The immunohistochemical characterization of the isolated T-I cells has been described previously [15]. Briefly, before plating, 95.4% of cells stained positive for a mesenchymal marker, vimentin; 7.6% of cells stained positive for an epithelial marker, cytokeratin; and 2.2% of cells stained positive for an endothelial marker, factor VIII. Comparable findings were observed at the end of the culture period. Furthermore, morphologic characteristics of the cultured cells were consistent with stromal cells and distinctly different from granulosa cells.
DNA Synthesis Determination
Radiolabeled [3H]thymidine (1 µCi/well) was added to cultured T-I cells during the last 24 h of culture. At the end of the culture period, the cells were harvested using a multiwell cell harvester (PHD Harvester, Model 290; Cambridge Technology, Inc., Watertown, MA). Radioactivity was measured in a liquid scintillation counter, SL 4000 (Intertechnique, Fairfield, NJ). Each treatment was carried out in at least 6 replicates.
Cell Counting, Identification of Steroidogenically Active Cells
T-I cells were cultured in 24-well plates; each treatment was carried out at least in triplicate. At the end of the culture period, the cells were washed with PBS. Trypsin-EDTA (0.05% and 0.02%, respectively; 0.3 ml/2 cm2) solution was dispensed into culture wells to completely cover the monolayer of cells, and the culture dish was placed at 37ÅC for 2–3 min. When the cells were in suspension and appeared rounded, McCoy's 5a medium was added to inhibit trypsin activity. Subsequently, the T-I cells were washed with PBS and fixed in 1% paraformaldehyde for 20 min. Steroidogenically active T-I cells were identified histochemically (Fig. 1) by detection of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity as described previously [20, 21]. The number of stained cells (steroidogenically active) and nonstained cells (steroidogenically inactive) was determined using a hemocytometer by counting 10 squares from each sample. Before cell counting, the samples were randomized, and the observer counting the cells was blinded to the treatment.
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Separation of Cells According to the Phase of the Cell Cycle
T-I cells were cultured in 6-well plastic plates with or without PDGF and IGF-I for 24 h (n = 5 for each treatment). Separation of the cells according to the phase of the cell cycle was performed using a supravital staining with a DNA binding dye (Hoechst 33342) in combination with the membrane potential modifying fluorochrome DiOC5 [22]. During the last hour of culture, T-I cells were incubated with Hoechst 33342 (5 µg/ml) and DiOC5 (0.3 µg/ml). Subsequently, the cells were trypsinized (as described above) and reconstituted in McCoy's 5a medium in the presence of Hoechst 33342 (5 µg/ml) and DiOC5 (0.3 µg/ml). Cell sorting was carried out using a FACS Vantage flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, CA). The stained cells were excited at 351–363 nm, and their fluorescence was observed between 440 and 480 nm. The cells were sorted according to their DNA content into two populations: proliferating (in S and G2/M phases of the cell cycle) or resting (in G0 and G1 phases of the cell cycle). Proliferating and resting populations of cells were then evaluated histochemically for the presence of 3ß-HSD activity (described above).
Statistical Analysis
Results are presented as the mean ± SEM. Comparisons between means were performed using ANOVA followed by post-hoc comparisons of individual means. Differences were considered significant at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA synthesis was evaluated by quantifying [3H]thymidine incorporation. PDGF alone (0.1–1 nM) produced a dose-dependent increase in DNA synthesis by 30–136% above control levels (Fig. 2). IGF-I alone (10 nM) increased DNA synthesis by 56% above the control. When the cells were exposed simultaneously to IGF-I (10 nM) and PDGF (0.1–1 nM), the radiolabeled thymidine incorporation increased by 108–214% above control levels, indicating that PDGF and IGF-I produced a near additive effect on DNA synthesis.
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The effect of the neutralizing antibody against PDGF (Ab-PDGF; 10 ng/ml) on DNA synthesis is presented in Figure 3. Basal thymidine incorporation was inhibited by 17%; furthermore, Ab-PDGF entirely neutralized the effects of the highest dose of PDGF (1 nM) on the DNA synthesis. In contrast, Ab-PDGF had no effect on the DNA synthesis in the presence of IGF-I.
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Determination of the Number of Steroidogenically Active and Inactive T-I Cells
Figure 4 presents the result of cell counting at the end of a 48-h culture. PDGF (1 nM) increased the total number of cells by 43% above the control; this effect was due to an increase in the population of steroidogenically inactive cells (by 47%) without significant change in the number of steroidogenically active cells. A distinctly different pattern was observed among T-I cells cultured in the presence of IGF-I (10 nM). IGF-I increased the total cell count by 64%; this effect was predominantly due to an increase in the number of steroidogenically active cells (by 163%); in addition, the number of steroidogenically inactive cells was also increased by 40%.
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Identification of Steroidogenic Activity among Proliferating and Resting Cells
Table 1 summarizes the effects of PDGF and IGF-I on the proportion of steroidogenically active cells among proliferating cells (in S and G2/M phases of the cell cycle) and among resting cells (in G0 and G1 phases of the cell cycle). In the absence of growth factors, the percentage of steroidogenically active cells was 35% lower among proliferating than resting cells. In the presence of PDGF (1 nM), the proportion of steroidogenically active cells was decreased among both proliferating and resting cells (by 43% and 16%, respectively). In contrast, in the presence of IGF-I (10 nM), the proportion of steroidogenically active cells increased among proliferating cells by 56%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The observation that steroidogenically active cells divide indicates that the presence of 3ß-HSD activity does not represent terminal differentiation in the traditional sense. Thus, while the development of steroidogenic activity may be viewed as a part of a continuum of the process of differentiation, it does not preclude mitotic activity. The findings of this study indicate, however, that differentiation of T-I cells, defined as the presence of steroidogenic activity, is associated with decreased proliferative activity. Under basal conditions in the absence of exogenous growth factors, proliferation among cells possessing 3ß-HSD activity was lower than proliferation among presumably less differentiated steroidogenically inactive cells. Exposure of the cells to IGF-I resulted in an overall increase in the number of steroidogenically active cells and a rise in the proportion of steroidogenically active cells in S and G2/M phases of the cell cycle. These findings suggest that the low basal proliferation of cells possessing 3ß-HSD activity may be selectively modified; in other words, the decrease of proliferative activity ascribed to differentiation may be reversed by an appropriate stimulus. Alternatively, in order to explain some of the above findings, one may postulate that IGF-I induced first proliferation and subsequently differentiation; this interpretation, however, does not explain the results obtained from flow cytometry experiments demonstrating that IGF-I increased the proportion of 3ß-HSD positive cells among actively dividing cells.
In contrast to IGF-I, PDGF preferentially stimulated proliferation of steroidogenically inactive cells and consequently decreased the proportion of cells with 3ß-HSD activity among proliferating cells. Thus, individual growth factors selectively stimulate separate populations of T-I cells. Such divergent actions of IGF-I and PDGF may provide mechanisms for selective development of ovarian stroma, theca externa, and theca interna. All these tissues contribute to ovarian structural integrity; however, stroma and theca externa possess relatively little steroidogenic activity, while theca interna is highly active and plays a major role in providing granulosa cells with aromatizable androgens. Thus, it is tempting to speculate that growth factors such as PDGF stimulate the growth of stroma and theca externa, while agents such as IGF-I stimulate development of theca interna.
Selective stimulation of proliferation of steroidogenically active and inactive cells by IGF-I and PDGF, respectively, indicates that these growth factors act independently of each other. Results of experiments with neutralizing antibodies against PDGF (Fig. 3) also indicate that IGF actions were independent of PDGF. Further evidence for independent activities of IGF-I and PDGF is provided by observations that proliferation was induced by either of these growth factors alone, and that a combination of IGF-I and PDGF had an additive effect. Thus, a traditional model postulating that proliferation requires both PDGF acting as a competence factor and IGF-I acting as a progression factor [17, 18] may not be strictly applicable to T-I cells. Indeed, it is apparent that effects of IGF-I and PDGF on proliferation are cell-specific. Thus, for example, in some systems, PDGF may stimulate DNA synthesis during both the competence and progression phases, while in other systems PDGF may be neither a competence nor a progression factor [23, 24].
The present observations also offer an interesting perspective on differentiation of the T-I compartment as a whole versus differentiation of individual T-I cells. Differentiation of the T-I compartment may be due not only to an increase in steroidogenesis by individual cells but be also a consequence of an increase in the number of steroidogenically active cells. In the latter case, steroidogenic activity of individual cells may remain unchanged, yet the steroidogenic output by the whole T-I compartment may increase. Notably, previous studies by Magoffin and Weitsman [13] revealed that IGF-I stimulated the expression of 3ß-HSD mRNA in cultures of T-I cells. In light of the present findings, it is likely that IGF-I increases 3ß-HSD expression, at least in part, by increasing the number of cells with steroidogenic activity (i.e., expressing 3ß-HSD).
In this study, T-I cells were obtained from estradiol-treated immature rats. This model provided ovaries with multiple medium-size antral follicles and actively dividing T-I cells. However, in vivo exposure to estradiol may affect a variety of receptors and thus alter endocrine and paracrine responsiveness of the cells. Another important limitation of this study is the possibility of a contamination of T-I cells by other cell types. To examine this possibility, the purity of the T-I cell preparation has been assessed with the aid of immunocytochemistry and morphologic examination of plated cells [15]. Significant contamination of T-I cells with endothelial and epithelial cells is unlikely, because of minimal immunostaining for factor VIII and cytokeratin. Contamination with leukocytes and monocytes was not studied; however, the perfusion of the animals with saline enabled removal of the bulk of blood cells. Finally, significant contamination with granulosa cells, while possible, is unlikely, since the morphologic appearance of the cultured cells is distinctly different from that of granulosa cells; furthermore, the isolation protocol used in this study has been thoroughly evaluated and has been shown to yield a T-I cell preparation nearly free of granulosa cells [25].
Another potential source of contamination of the cultures is BSA, which may contain growth factors and cytokines. However, such contamination would not be likely to alter the findings of this study since all cultures were carried out in the presence of the same concentrations of BSA (1 mg/ml).
In summary, we postulate that PDGF and IGF-I induce selective proliferation of specialized sub-populations of T-I cells. Such actions may represent mechanisms leading to selective development of ovarian stroma, theca externa, and theca interna.
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FOOTNOTES |
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2 Correspondence: Antoni J. Duleba, M.D., Yale University School of Medicine, Department of Obstetrics and Gynecology, 333 Cedar Street, New Haven, CT 06520. FAX: 203 785 7134; antoni.duleba{at}qm.yale.edu
Accepted: October 5, 1998.
Received: August 20, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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