Molecular Human Reproduction, Vol. 5, No. 3, 193-198,
March 1999
© 1999 European Society of Human Reproduction and Embryology
Divergent mechanisms regulate proliferation/survival and steroidogenesis of theca–interstitial cells
1 Yale University School of Medicine, Department of Obstetrics and Gynecology, 333 Cedar Street, New Haven, CT 06520–8063, and 2 Karol Marcinkowski University School of Medical Sciences, Department of Gynecology and Obstetrics, ul. Polna 33, Poznan, Poland
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Abstract |
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Luteinizing hormone (LH) and insulin-like growth factor I (IGF-I) are recognized as major regulators of ovarian theca–interstitial (T-I) function. This study was designed to compare the effects of LH and IGF-I on T-I proliferation and steroidogenesis. Purified rat T-I cells were cultured in chemically-defined media. DNA synthesis was evaluated by a radiolabelled thymidine incorporation assay. The cells were also directly counted. Progesterone production was assessed using a specific radioimmunoassay. DNA synthesis of T-I cells was stimulated by IGF-I (10 nM) but modestly inhibited by LH (100 ng/ml). The inhibitory effect of LH was mimicked by 8Br-cAMP (10–4 to 10–3 M); forskolin (10–5 M), cholera toxin (10 ng/ml) and 3-isobutyl-methyl-xanthine (10–5 M). Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (10–7 M) had no significant effect on DNA synthesis. Furthermore, DNA synthesis was not affected by testosterone (10–10 to 10–9M) or progesterone (10–9 to 10–8 M). Accumulation of progesterone was co-operatively stimulated by LH and IGF-I. These results suggest that LH-induced inhibition of T-I proliferation and/or survival is mediated via the cAMP system. IGF-I may be viewed as a co-gonadotrophin with respect to steroidogenesis but not with respect to proliferation/survival. The divergence of the effects on proliferation/survival versus steroidogenesis underscores the complexity of the interactions between LH and IGF-I signalling pathways.
IGF-I/LH/proliferation/steroidogenesis/theca–interstitial cells
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Introduction |
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Control of proliferation and differentiation of theca–interstitial (T-I) cells is essential for appropriate ovarian development and homeostasis. In the mature ovary, homeostasis of the T-I compartment is a dynamic, orderly development. Maintenance and regression of follicles require mechanisms regulating growth and steroidogenesis of the follicular wall.
Luteinizing hormone (LH) and insulin-like growth factor I (IGF-I) are well recognized as major regulators of T-I function. LH stimulates theca cells to produce androgens in a broad range of species including rat, rabbit, cattle, and humans (Erickson and Ryan, 1976
; Fortune and Armstrong, 1977; McNatty et al., 1984; Bergh et al., 1993). LH also maintains progesterone production (Bogovich et al., 1986). The above effects of LH are due to the stimulation of several enzymatic activities: cholesterol side-chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and 17
-hydroxylase (P45017) (Bogovich and Richards, 1982; Magoffin et al., 1990; Magoffin and Weitsman, 1993a,b). Actions of LH on steroidogenesis are mimicked by stimulation of the cAMP system (Erickson and Ryan, 1976; Richards et al., 1986).
There is growing evidence that IGF-I mimics and/or modulates actions of gonadotrophins (Erickson et al., 1994; Kol et al., 1997). In particular, IGF-I synergistically augments LH stimulation of androgen production by T-I cells (Cara and Rosenfield, 1988). This effect may be partly due to IGF-I enhancement of LH binding capacity (Cara et al., 1990; Magoffin and Weitsman, 1994). However, some actions of IGF-I on steroidogenesis are independent of LH. For example, IGF-I alone stimulates both P450scc and 3ß-HSD in purified T-I cells of hypophysectomized rats (Magoffin et al., 1990; Magoffin and Weitsman, 1993b). In contrast, IGF-I stimulates expression of P45017 mRNA only in the presence of LH (Magoffin and Weitsman, 1993a). Thus, IGF-I acts on T-I steroidogenesis by both augmenting and mimicking the actions of LH.
Function of the T-I compartment depends not only on its steroidogenic activity, but also on the number of cells. We have recently demonstrated that IGF-I is a potent stimulator of T-I cell proliferation leading to an increase in both the number and the proportion of steroidogenically active T-I cells (Duleba et al., 1997). The present study was designed to characterize the role of LH in the regulation of T-I cell proliferation and to investigate the potential mechanisms mediating these actions. The effects of LH and IGF-I on T-I proliferation were compared with the actions on steroidogenesis. To our knowledge, this is the first report demonstrating that LH decreases basal proliferation of T-I cells and that this effect is mimicked by stimulation of the cAMP system.
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Materials
The following materials were purchased from Sigma Chemical Co (St Louis, MO, USA): Medium-199 with Hank's balanced salt solution (HBSS; x10), McCoy's 5a medium (modified, without serum), L-glutamine, bovine serum albumin (BSA), trypsin–EDTA (0.05%/0.02%), Nitroblue Tetrazolium, 5ß-androstan-3ß-ol-17-one, ß-NAD+, sesame oil, Percoll, progesterone, testosterone, cholera toxin, forskolin, 8-bromo-cAMP, 3-isobutyl-methyl-xanthine, phorbol 12-myristate 13-acetate and human recombinant IGF-I. Collagenase type I (Clostridium histolyticum, CLS1; 146 IU/mg) and DNase I (bovine pancreas; 2298 IU/mg) were obtained from Worthington Biochemical Co (Freehold, NY, USA). RU 486 was obtained from Roussel UCLAF (Romainville, France). The following materials were purchased from Grand Island Biological Co (Grand Island, NY, USA): Trypan Blue stain (0.4%; wt/vol.), antibiotic-antimycotic preparation (penicillin, 10 000 IU/ml; streptomycin, 10 000 µg/ml; amphotericin B, 25 µg/ml), and Dulbecco's phosphate-buffered saline (PBS, x1, pH = 7.2, without MgCl2 and CaCl2). HEPES was purchased from American Bioanalytical (Natick, MA, USA). Radiolabelled [3H]-thymidine, and [3H]-progesterone were purchased from Amersham Life Science Inc. (Arlington Heights, IL, USA). Ovine LH (o-LH-26) was kindly donated by National Hormone and Pituitary Program (NIDDK; Bethesda, MD, USA). Contamination of o-LH-26 with other anterior pituitary hormones was as follows: growth hormone (GH) <0.1%, thyroid stimulating hormone (TSH) <0.5%, follicle stimulating hormone (FSH) <0.5%, and prolactin (PRL) <0.1%.
Animals
Immature (25 days old) female Sprague–Dawley rats were obtained from Taconic Farms (Germantown, NY, USA) and housed with a 12 h light:12 h dark photoperiod in an air-conditioned environment. Standard rat chow and water were given ad libitum. Starting at day 28 of age the animals were injected with 17ß-oestradiol (1 mg/0.3 ml sesame oil s.c.) daily for 3 days in order to stimulate ovarian development. On the morning following the last injection (day 31 of age) the animals were anaesthetized 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.
Isolation of theca–interstitial cells and cell cultures
Ovaries were dissected, and T-I cells were isolated and purified as described previously (Duleba et al., 1997). The cells were counted using a haemocytometer and viability was determined using Trypan Blue Stain 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) and with or without insulin and IGF. Cultures were incubated at 37°C under an atmosphere of 5% CO2 in humidified air in 24-well (Falcon, Becton Dickinson Labware, Lincoln Park, NJ, USA) or 96-well plastic plates (Corning Glass Works, Corning, NY) for up to 96 h. The final concentration of T-I cells was 350x103 cells/ml in 24-well plates and 35x103 cells/0.25 ml in 96-well plates. At the end of the culture period, the viability of the attached cells was in the 90–95% range. Immunohistochemical evaluation of the cells was performed before the plating and at the end of the culture period. Both cell populations had comparable staining to vimentin (90–95%), cytokeratin (5–10%), and factor VIII (<5%). Details of the immunohistochemical methodology were presented previously (Duleba et al., 1997).
DNA synthesis
Radiolabelled [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, USA). Radioactivity was measured in a liquid scintillation counter, SL 4000 (Intertechnique, Fairfield, NJ, USA). Each treatment was carried out in at least six replicates.
Cell counting, identification of steroidogenically active cells, radioimmunoassays
T-I cells were cultured in 24-well plates; each treatment was carried out in at least triplicate. At the end of the culture period, the cells were washed with PBS (x1, pH = 7.2). 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 37°C for 2–3 min. When 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 (x1, pH = 7.2) and fixed in 1% paraformaldehyde for 20 min. Steroidogenically active T-I cells were identified histochemically by detection of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity as described previously (Hild-Petito et al., 1989; Bao et al., 1995; Duleba et al., 1997). Briefly, T-I cells were reconstituted in histochemical staining solution (PBS, x1, supplemented with 0.1% BSA, 1.5 mM ß-NAD+, 0.25 mM Nitroblue Tetrazolium, and 0.2 mM 5ß-androstan-3ß-ol-17-one) and incubated overnight at 37°C. Under these conditions, 5ß-androstan-3ß-ol-17-one served as a substrate to 3ß-HSD; blue staining of the cells was the effect of the subsequent redox reaction with Nitroblue Tetrazolium. The number of stained cells (steroidogenically active) and non-stained cells (steroidogenically inactive) was determined using a haemocytometer. Prior to cell counting, the samples were randomized and the observer counting the cells was blinded to the treatment. Concentrations of progesterone in the spent culture media was carried out with the aid of specific radioimmunoassay as described previously (Orczyk et al., 1979).
Statistical analysis
Results are presented as the mean ± SEM unless stated otherwise. Comparisons between the means were performed using analysis of variance (ANOVA) followed by post-hoc testing using, when appropriate, Bonferroni correction. Differences were considered to be statistically significant when P < 0.05.
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Results |
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Effects of LH and IGF-I on DNA synthesis
DNA synthesis was estimated by the [3H]-thymidine incorporation assay. Time-course of the effects of LH on DNA synthesis is presented in Figure 1
. T-I cells were incubated in serum-free medium with or without LH (100 ng/ml) for up to 72 h. In the presence of LH the synthesis of DNA was modestly decreased (by 13–25%) at all tested time points. This inhibitory effect of LH was statistically significant (P < 0.05) accounting for the effect of time by two-way analysis of variance. Since the incorporation of thymidine was minimal at 24 h and significantly greater thereafter, subsequent experiments were performed during 48–72 h incubations. We have previously demonstrated that this is also the optimal time for detection of the effects of IGF-I (Duleba et al., 1997).
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Determination of DNA synthesis in the presence of LH (1–100 ng/ml) and/or IGF-I (10 nM) is presented in Figure 2
. LH alone induced a modest suppression of DNA synthesis at 100 ng/ml (P < 0.05); at lower doses, LH had no significant effect. In contrast, IGF-I (10 nM) significantly increased DNA synthesis by 3.2-fold at 48 h (P < 0.001) and by 2.1-fold by 72 h (P < 0.001). LH had no significant effect on IGF-I-induced DNA synthesis.
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Role of cyclic AMP and protein kinase C systems on DNA synthesis
Actions of LH on T-I cells may be mediated via transduction pathways involving cyclic AMP (cAMP) and protein kinase C (PKC) systems (Rajkumar et al., 1991
; Shimamoto et al., 1993; Davis, 1994). In order to evaluate the role of these systems in the regulation of DNA synthesis, T-I cells were cultured for up to 72 h with or without 8-bromo-cAMP (8Br-cAMP, 10–5 to 10–3 M); forskolin (10–5 M), cholera toxin (10 ng/ml), 3-isobutyl-methyl-xanthine (IBMX, 10–5 M), and phorbol 12-myristate 13-acetate (PMA, 10–7M). The effects of these agents on thymidine incorporation are presented in Figure 3. DNA synthesis was significantly decreased by 8Br-cAMP in a dose-dependent fashion with the highest dose (10–3M) producing inhibition by 88% below control level (P < 0.001). A significant decrease of DNA synthesis was also induced by stimulation of cAMP accumulation using forskolin (inhibition by 60%; P < 0.0001) and cholera toxin (inhibition by 44%; P < 0.05). Similarly, blocking of cAMP-phosphodiesterase activity with IBMX led to inhibition of thymidine incorporation by 44% (P < 0.05). In contrast, stimulation of PKC by PMA had no significant effect on DNA synthesis.
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Regulation of steroidogenesis
Effects of LH, IGF-I, and 8Br-cAMP on progesterone production by T-I cells are presented in Figure 4
. At 96 h, cumulative production of progesterone was significantly increased by LH and 8Br-cAMP (respectively by 77 and 367% above control level; P < 0.05). IGF-I alone had no significant effect on progesterone accumulation; however, a combination of LH and IGF-I stimulated progesterone production significantly (P < 0.05) above the level attained in the presence of LH alone (by 248% above control level).
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DNA synthesis in the presence of progesterone and testosterone
It is possible that LH and stimulation of the cAMP accumulation may affect DNA synthesis indirectly, via effects induced by steroids produced by T-I cells. To address this possibility, DNA synthesis by T-I cells was evaluated in the presence or absence of progesterone (10–9 to 10–8 M), progesterone antagonist RU486 (10–6 M), and testosterone (10–10 to 10–9 M). DNA synthesis was not significantly affected by any of these treatments (results not shown).
Determination of the T-I cell number and the presence of 3ß-hydroxysteroid dehydrogenase activity
The effect of LH and 8Br-cAMP on T-I cell proliferation was re-evaluated by directly counting the steroidogenically active cells (3ß-HSD-positive) and steroidogenically inactive cells (3ß-HSD-negative). Results of a representative experiment are presented in Figure 5. In the presence of LH, there was a trend towards a modest decrease in the total cell count (by 9%) due mostly to a 33% decline in the number of steroidogenically active cells. These trends did not reach statistical significance. 8Br-cAMP induced a significant decline in the total number of T-I cells (by 53% below control, P < 0.01); this effect consisted of a comparable decline in the number of steroidogenically active (by 54%, P < 0.05) and steroidogenically inactive cells (by 53%, P < 0.01). Notably, only 2–4% of plated cells were recovered at the end of the culture period. Repeated experiments using both Coulter counter and direct cell counting with haemocytometer confirmed that cultures in chemically-defined media were associated with loss of up to 98% of cells.
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Discussion |
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The present findings indicate that: (i) DNA synthesis of T-I cells is inhibited by LH and stimulated by IGF-I; (ii) the inhibitory effect of LH is mimicked by stimulation of the cAMP accumulation; and (iii) in contrast to the actions on DNA synthesis, LH and IGF-I cooperatively stimulate steroidogenesis.
The above findings may be relevant to a better understanding of both ovarian physiology and pathophysiology. Regulation of the size of stromal–thecal compartment is essential to the maintenance of ovarian structural integrity and the control of follicular development and atresia. Thus, LH may be viewed as an agent protecting the T-I compartment from hyperplasia, possibly by inducing terminal differentiation of target cells.
In view of the low recovery of cells in serum-free cultures, it is likely that changes in the thymidine incorporation may be due, at least in part, to the alterations of T-I cell survival. LH and cAMP accumulation may decrease cell survival, while IGF-I may have an opposite effect. Consequently, the observed effects on the DNA synthesis may represent a net result of alterations of the cell survival and proliferation. The low recovery of cells at the end of the 72 h culture also raises concerns as to whether the effects of treatments were reliably detected and whether the cells remaining at the end of the culture period were representative of plated T-I cells. The reliability of the observed effects is supported by comparable findings from two independent methodologies: determination of thymidine incorporation and direct cell counting. Furthermore, immunohistochemical characterization was carried out on the cells before plating and at the end of the culture period. Comparable percentages of both cell populations stained positive for vimentin, cytokeratin, and factor VIII, indicating that the cells present at the end of the culture represented the plated cells. The above comments notwithstanding, the study conditions resulted in a selection of cells able to survive and proliferate in serum-free conditions.
Previous literature on the actions of LH on proliferation of T-I cells is scant. In 1978 Rao et al. found that LH may stimulate a limited proliferation of these cells for up to 48 h (Rao et al., 1978). The discrepancy in the conclusions of that report and the present observations may be due to important differences in the design and the execution of these studies. First, in the study of Rao et al. (1978), LH was administered in vivo and only after sequentially exposing the animals to oestradiol and FSH; the effects of LH were inferred on the basis of the temporal relationship between the administration of LH and proliferation. In such a model, the observed changes in the proliferation of T-I cells may have reflected an interaction between several hormones or a delayed response to oestradiol and FSH, rather than a direct effect of LH. In addition, T-I preparations were not purified and were likely to be significantly contaminated by granulosa cells. In contrast, in the present study, actions of LH were evaluated in vitro and in direct comparison to control cultures. Furthermore, T-I cells were highly purified (Duleba et al., 1997). The effects of LH may be also species-specific. Onagbesan et al. (1994) observed that LH had a modest stimulatory effect on proliferation of theca interstitial cells of the domestic hen. Notably, enhancement of proliferation was demonstrated only in some follicles and, in agreement with our study, LH had no effect on proliferation in the presence of IGF-I.
The present study has shown that actions of LH on proliferation/survival of T-I cells are mimicked by the cAMP. This observation underscores the complex and tissue dependent role of cAMP in regulation of proliferation. Early reports have documented negative control, whereby cAMP inhibited a proliferation in broad range of cells including adrenal cells, fibroblasts, and luteal cells (Frank, 1972; Gospodarowicz and Gospodarowicz, 1975; Ramachandran and Suyama, 1975). More recent studies have demonstrated that cAMP enhances or initiates proliferation in many systems such as Sertoli cell and thyroid cell cultures (Dorrington et al., 1974; Takahashi et al., 1990). Importantly, actions of cAMP on proliferation may be biphasic with stimulation at low concentrations and inhibition at high concentrations; such effects have been observed in rat granulosa cells (Bley et al., 1992). In the present study, we found no evidence of biphasic effects and inhibition of DNA synthesis was observed even at relatively low concentrations of 8Br-cAMP.
Actions of LH may be mediated not only by cAMP but also by other pathways, for example via PKC (Rajkumar et al., 1991; Shimamoto et al., 1993; Davis, 1994). In the present study, stimulation of PKC with PMA had no effect on T-I proliferation. This observation is in agreement with Hofeditz et al. (1988) who found that stimulators of PKC had no effect on the total DNA and protein concentrations in cultures of rat T-I cells. Another potential pathway mediating LH actions may involve indirect effects of LH induced via stimulation of the production of ovarian steroids such as testosterone and progesterone which, in turn, may affect function of T-I cells. In this study, we found no evidence of an involvement of testosterone or progesterone in modulation of T-I proliferation. The lack of testosterone-mediated effects correlates well with previously reported absence of thecal hyperplasia in patients treated with large doses of exogenous androgens (Amirikia et al., 1986).
The interactions between LH and IGF-I probably play a significant role in the pathophysiology of the polycystic ovary syndrome (PCOS), one of the most common endocrinopathies affecting women of reproductive age (Yen et al., 1970; Suikkari et al., 1989; Iwashita et al., 1990; Homburg et al., 1992). PCOS is associated with an elevation of LH (Yen et al., 1970) and free-bioavailable IGF-I (Iwashita et al., 1990; Homburg et al., 1992); the latter effect is probably due to a decrease in the levels of IGF-binding protein-1 (Suikkari et al., 1989; Homburg et al., 1992). The prominent clinical features of PCOS include hyperandrogenism and ovarian thecal and stromal hyperplasia (Adams et al., 1985; Clement, 1987; Damjanov, 1993). While one has to be cautious in interpreting human pathophysiology on the basis of rodent studies, it is tempting to speculate that synergistic actions of LH and IGF-I on steroidogenesis may explain hyperandrogenism, while elevations of free IGF-I may exert the dominant influence leading to thecal and stromal hyperplasia. In agreement with this hypothesis, in the present study, antiproliferative actions of LH were evident only in the absence of IGF-I.
In summary, we have demonstrated that proliferation and/or survival of T-I cells is inhibited by LH and stimulated by IGF-I; in contrast, steroidogenesis is co-operatively stimulated by both LH and IGF-I. Thus, IGF-I may be viewed as a co-gonadotrophin with respect to steroidogenesis but not with respect to the regulation of the T-I cell number. This divergence of the effects on proliferation/survival versus steroidogenesis underscores the complexity of the interactions between LH and IGF-I signalling pathways.
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Acknowledgments |
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We would like to acknowledge that ovine LH (o-LH-26) was kindly donated by National Hormone and Pituitary Program, NIDDK.
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Notes |
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3 To whom correspondence should be addressed
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References |
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Submitted on May 14, 1998; accepted on November 30, 1998.
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