Molecular Human Reproduction, Vol. 5, No. 11, 1003-1010,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular endocrinology |
Insulin and insulin-like growth factor-I and- II modulate human granulosa–lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression
1 Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia, PA, 19104, 2 Department of Cell and Molecular Physiology, Pennsylvania State University, Milton S .Hershey Medical Center, Hershey, PA 17033, USA
Abstract
Insulin and insulin-like growth factors (IGF)-I and -II stimulate granulosa cell steroidogenesis. Since steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis, the ability of insulin and IGF to modulate StAR protein and mRNA expression was examined in two human granulosa cell culture systems: (i) proliferating granulosa–lutein cells and (ii) luteinized-granulosa cells derived during in-vitro fertilization (IVF). In proliferating granulosa–lutein cells, IGF-I and IGF-II increased StAR protein ~4–5-fold, while insulin and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) increased amounts of StAR protein 2.5- and 6-fold respectively. A combination of IGFs/insulin and 8-Br-cAMP increased StAR 7–9-fold. Luteinized granulosa cells also had increased StAR expression after treatment with IGF-I (2.8-fold), IGF-II (3-fold), insulin (2.5-fold) and 8-Br-cAMP (4.5-fold). Progesterone production generally followed a pattern similar to StAR protein in both cell systems. In proliferating granulosa–lutein cells, both IGF-I and insulin increased StAR mRNA (3-fold) and 8-Br-cAMP increased StAR mRNA 4-fold, whereas a combination of IGF-I and 8-Br-cAMP had an additive effect on StAR mRNA expression (7-fold). Transient transfection of proliferating granulosa–lutein cells with StAR promoter-luciferase reporter constructs demonstrated that IGF-I, IGF-II, and insulin had no effect on the StAR promoter activity, while 8-Br-cAMP stimulated StAR promoter function. The results indicate that: (i) IGFs and insulin stimulate StAR mRNA and protein expression in human granulosa–lutein cells; (ii) IGF-I and 8-Br-cAMP have an additive effect on StAR gene expression; and (iii) IGF-I increases StAR mRNA and protein by a mechanism that does not involve activation of the proximal StAR gene promoter.
granulosa cell/human steroidogenesis/insulin like growth factors/steroidogenic acute regulatory protein
Introduction
Thecal, granulosa and luteal cell steroidogenesis is dependent on pituitary-derived follicle stimulating hormone (FSH) and luteinizing hormone (LH), acting through the cAMP second messenger signalling system to co-ordinately regulate genes essential for hormone synthesis and normal follicular and luteal development. In addition to cAMP, the actions of FSH and LH are modified by a variety of growth factors/cytokines and hormones to achieve the mitogenic and steroidogenic effects within the follicle and corpus luteum respectively. Insulin and the insulin-like growth factors (IGF-I) and (IGF-II) are known to modulate ovarian steroidogenesis and mitotic activity in several species including the human (Adashi et al., 1985
; Guidice, 1992; Kol et al., 1997). These polypeptides stimulate the replication of a variety of cell types and appear to have autocrine/paracrine actions as well as classical endocrine functions (Jones and Clemmons, 1995).
The complete IGF system encompassing the IGF proteins, IGF receptors, IGF-binding proteins and the IGF-binding protein protease have been detected in human peri-ovulatory follicles (Hernandez et al., 1992; El-Roeiy et al., 1994; Mason et al., 1996; De Neubourg et al., 1998; Mimuro et al., 1998). Interestingly, human granulosa cells express IGF-I, IGF-II and insulin specific-receptors as well as IGF-II ligand, but lack IGF-I mRNA, even though IGF-I can be detected in follicular fluid, suggesting an extra-follicular source (van Dessel et al., 1996). It is thought that IGFs stimulate steroidogenesis through the IGF type I tyrosine kinase class of cell surface receptors, while insulin signals through its own receptor at physiological concentrations (Willis and Franks, 1995). The stimulatory effects of IGFs on steroidogenic cells have been attributed to increased cAMP generation (Adashi et al., 1988), enhanced uptake of lipoproteins, and stimulation of the activity and expression of several steroidogenic enzymes, including P450 side-chain cleavage and 3ß-hydroxysteroid dehydrogenase (de Moura et al., 1997). However, there is limited information regarding the action of IGFs and insulin as modulators of cholesterol transfer to the inner mitochondrial membrane, which is the rate-limiting step in steroid hormone production (Stocco and Clark, 1996). Steroidogenic acute regulatory protein (StAR) was shown to be the protein that stimulates the translocation of cholesterol from the sterol-rich outer mitochondrial membranes to the cholesterol-poor inner mitochondrial membranes where the P450 side chain cleavage system converts it into pregnenolone. Furthermore, the induction of StAR gene expression in luteinized human granulosa cells was shown to directly correlate with the ability of these cells to produce progesterone. In addition to transcriptional regulation, StAR function can also be modulated by cAMP-directed phosphorylation of serine 195 (Arakane et al., 1997). This post- or co-translational event may account, in part, for the acute regulation of steroid production by cAMP. The discovery of StAR delineated new potential mechanisms by which IGF-I, IGF-II and insulin could control steroidogenesis through increases in StAR protein concentrations and/or action via transcriptional/translation and/or post-translational mechanisms. In the present study, the ability of IGFs and insulin to regulate basal and cAMP-stimulated StAR mRNA expression and protein concentrations in human granulosa cells was examined. Furthermore, studies were carried out to elucidate the mechanisms by which IGF/insulin influence StAR gene expression.
Materials and methods
Cell culture
Two different human granulosa cell systems were employed: proliferating granulosa–lutein cells which afford sufficient numbers of cells to conduct studies on gene expression and promoter activity using transfection protocols, and luteinized granulosa cells collected from women undergoing in-vitro fertilization (IVF), which do not replicate in culture but have greater steroidogenic capabilities than the proliferating granulosa–lutein cells. Two different cell preparations were studied in order to establish the generality of the actions of IGFs and insulin on StAR expression in granulosa–lutein cells. Proliferating human granulosa–lutein cells were prepared and cultured as previously described (McAllister et al., 1990). Briefly, human granulosa cells were expanded on fibronectin-coated 100 mm tissue culture dishes in Dulbecco's modified Eagle's medium (DMEM) + Ham's F12 medium (1:1; DMEM/F12) supplemented with 10% heat-treated fetal calf serum (FCS) and antibiotics. Cultures were maintained in an atmosphere of 95% air-5% CO2 at 37°C. Once granulosa cells reached 60–80% confluence they were cultured in serum-free DMEM/F12 + 0.1% bovine serum albumin (BSA), 100 µg/ml transferrin and 20 nmol/l selenium for 2 days before treatment. Fresh media (serum-free) containing the maximal stimulatory doses of 1 mmol/l 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP; Sigma, St Louis, MO, USA), 50 ng/ml recombinant human IGF-I, (Roche/Boehringer Mannheim, Indianapolis, IN, USA) or recombinant human IGF-II (Roche/Boehringer Mannheim), 20 nmol/l recombinant human insulin (Roche/Boehringer Mannheim) or combinations of the stimulatory peptides plus 8-Br-cAMP were then added to cells for a 24 h period. Preliminary dose response curves indicated that 50 ng/ml of IGF-I or IGF-II elicited a maximal stimulatory response. After the 24 h incubation, the media were collected and frozen for subsequent progesterone determinations and the cells were processed for Northern blot and Western blot analyses.
Primary cultures of human granulosa–lutein cells were established from cells collected from patients undergoing oocyte retrieval following a standard follicular hyperstimulation protocol (Makrigiannakis et al., 1999). Briefly, patients received a gonadotrophin-releasing hormone (GnRH) agonist (Lupron, TAP Pharmaceuticals, Deerfield, IL, USA) for pituitary suppression and recombinant FSH (Gonal F; Serono, Randolph, MA, USA) for follicular recruitment followed by a single dose of human chorionic gonadotrophin (HCG; 10 000 IU) 36 h before oocyte retrieval. Cells from individual patients were washed in DMEM/F12 and contaminating red blood cells were removed with a Ficoll gradient. The cell pellet was resuspended in DMEM/F12 +10% FCS (Organon Teknika, Durham, NC, USA) and the cells were plated for 20 min, followed by aspiration of the non-adherent cells, centrifugation at 400 g for 30 min and re-suspension of the cell pellet in DMEM/F12 + 0.1% BSA. This procedure removes macrophages (adherent cells) and results in a 95% pure population of human granulosa cells. Cells were then plated and cultured on fibronectin-coated 6-well plates. Cells and media were then collected 24 h after plating (day 1) or cells were cultured for 5 days before treatments were initiated. After 24 h incubation with 8-Br-cAMP, IGF-I, IGF-II or insulin, the media was collected and the cells were processed for Western blot analysis.
Northern blot analysis
The effect of the IGFs and insulin in the presence or absence of 8-Br-cAMP on StAR mRNA was evaluated in proliferating human granulosa cells. Briefly, total RNA was isolated from cells as previously described (Kiriakidou et al, 1996), and resolved (10 µg/lane) on 1% denaturing agarose gels and blotted onto nylon membranes (Hybond-N+; Amersham Life Sciences Inc., Arlington Heights, IL, USA) by capillary transfer. Blots were probed with radiolabelled human StAR cDNA, stripped and reprobed with a labelled 28S RNA cDNA probe in order to assess RNA loading. Hybridization signals were visualized on a Storm Phosphor Imager and relative amounts of StAR mRNA and 28S rRNA cDNA were determined with the ImageQuant 1.11 software (Molecular Dynamics, Sunnyvale, CA, USA).
Western blot analysis
Protein extracts from proliferating and primary granulosa cells were produced as previously described (Arakane et al., 1997). Equal amounts of protein (15 µg) as determined by the BioRad dye binding assay were loaded onto sodium dodecyl sulphate–polyacrylamide gels (10%) for electrophoresis. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Hybridon; Millipore Co, Bedford, MA, USA) for detection with antihuman-StAR antibody. The rabbit polyclonal antibody was produced against recombinant human StAR protein as described previously (Arakane et al., 1997). The detection of bound anti-StAR antibody was carried out using the Vistra ECF Western Blotting kit (Amersham) and the chemifluorescence signal was detected on the Storm PhosphoImager using the blue fluorescence/chemifluorescence mode.
Cell transfection and luciferase assay
Proliferating human granulosa cells were plated onto 12-well plates at a density of 9x105 cells/well on day 0 in DMEM/F12 + 10% FCS. On the afternoon of day 1, cells were transfected with FUGENE-6 (1.5 µl; Roche/Boehringer Mannheim) and 500 ng of the 1.3 kb human StAR promoter fused to the pGL2-luciferase reporter gene (Sugawara et al., 1997) and 100 ng of ß-galactosidase expression vector (pCMV-ßgal; Stratagene, La Jolla, CA, USA). Cells were left overnight in the medium (DMEM/F12 + 10% FCS + antibiotics) containing the FUGENE/DNA mixture. On day 2, cells were washed with serum-free DMEM/F12 medium and maintained in this medium for 24 h, before being exposed to IGFs or insulin in the presence or absence of 8-Br-cAMP for an additional 24 h at which time cells were harvested. The ß-galactosidase expression vector was used for normalization of luciferase data.
Luciferase and ß-galactosidase and progesterone assays
Luciferase activity was determined in a LUMAT LB 9507 luminometer (EG & G Berthold, Bad Wildbach, Germany) with Promega luciferin as substrate as described previously (Sugawara et al., 1997). ß-galactosidase activity was determined by standard colourimetric assays using 2-nitrophenyl-ß-galactopyranoside as substrate. Relative luciferase activity for each well was determined by dividing luciferase relative light units by the ß-galactosidase activity (A420). Progesterone determinations were performed using a kit obtained from Diagnostic Products (Los Angeles, CA, USA).
Statistical analysis
Northern and Western blotting experiments performed with proliferating human granulosa cells were repeated at least four times for each stimulatory peptide tested. A total of eight separate cultures were completed and the data is presented as mean ± SEM; the number of replicates for each treatment is indicated in the figure legends. Experiments with primary granulosa cells represent the means ± SEM studies conducted on cells collected from seven different patients. All data were analysed by analysis of variance (ANOVA), following the detection of a significant F-test (P < 0.05) differences among individual means were tested by paired Student t-tests. P < 0.05 was considered to be statistically significant.
Results
Figure 1 shows a Western blot analysis of StAR proteins from proliferating human granulosa–lutein cells treated with IGF-I, IGF-II or insulin in the absence or presence of 8-Br-cAMP. None of these treatments affected cell viability as assessed by light microscopic examination (e.g. changes in numbers of floating cells or cells with nuclear fragmentation). All treatments increased the amount of StAR protein over that found in the cells cultured under control conditions. A single immunoreactive band at 30 kDa representing the mature StAR protein was observed in all treatment groups: there was no evidence of the larger molecular weight form of StAR representing the preprotein in these blots.
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Figure 2A
depicts the results of the Western analysis and (Figure 2B) media progesterone concentrations for proliferating human granulosa–lutein cells. IGF-I augmented StAR protein concentrations 5-fold and insulin 2.5-fold over control values. Similarly, treatment with IGF-II increased StAR values 4-fold in two experiments (not statistically analysed; see Figure 1
). Interestingly, progesterone concentrations for IGF-I and insulin-treated cells were not significantly increased following the 24 h treatment of the proliferating human granulosa cells. 8-Br-cAMP at the dose of 1 mmol/l increased StAR protein values 6.5-fold and progesterone production 3-fold over control values. The combination of IGF-I and 8-Br-cAMP produced the greatest increase in StAR protein (9-fold) over control values, however, this increase was not significantly different from the cells treated with 8-Br-cAMP or IGF-I alone. IGF-I and insulin also failed to further stimulate progesterone secretion by proliferating granulosa cells treated simultaneously with 1mmol/l 8-Br-cAMP.
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Figure 3
shows a Western blot of StAR protein in primary cultures of IVF-derived luteinized granulosa cells 24 h (day 1) after collection and after 6 days of culture (control) and following a 24 h treatment with IGFs, insulin and 8-Br-cAMP. Figure 4A depicts the mean ± SEM for seven independent experiments examining the amount of StAR protein in luteinized granulosa cell cultures and Figure 4B shows progesterone accumulation in medium for the above treatments. Amounts of StAR protein and media progesterone concentrations were at their highest value 24 h after collection of the cells and had markedly decreased by the sixth day of culture (P < 0.05). Cells stimulated with IGF-I, IGF-II, insulin or 8-Br-cAMP had significantly increased (P < 0.05) amounts of StAR protein, compared with those observed in control cells. Production of progesterone followed a similar pattern (r = 0.83; P < 0.05) as that of the StAR protein in the luteinized granulosa cells, confirming the tight association between StAR protein and progesterone biosynthesis.
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Results of Northern blot analyses of StAR mRNA in proliferating human granulosa–lutein cells following treatment with IGF-I and insulin in the absence or presence of 8-Br-cAMP are shown in Figures 5A–C
. StAR mRNA (1.7 kb transcript) was up-regulated 2.5–3-fold (P < 0.05) in granulosa–lutein cells treated with IGF-I or insulin alone, when compared with control cells after a 24 h culture period. Treatment with 8-Br-cAMP increased the amount of StAR mRNA ~4-fold compared with the control cells (P < 0.05). Proliferating granulosa–lutein cells treated with IGF-I + 8-Br-cAMP contained 7-fold more StAR mRNA than control cells. The increase in StAR mRNA with the combination treatment was significantly higher than cells treated either with 8-Br-cAMP or IGF-I alone (P < 0.05), suggesting an additive effect on StAR mRNA expression. Cells treated with insulin + 8-Br-cAMP also displayed greater StAR mRNA expression than those treated with 8-Br-cAMP alone, but the difference did achieve statistical significance. A larger StAR mRNA (4.2 kb) transcript was observable, however, expression was very low (near background expression levels) and not analysable.
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To determine whether IGFs and insulin stimulate StAR gene transcription, the 1.3 kb and 235 bp StAR promoters fused to a luciferase reporter were transiently transfected into proliferating granulosa–lutein cells followed by 24 h of agonist treatment. Using the 1.3 kb StAR promoter construct, experiments revealed that IGF-I was unable to augment StAR promoter activity, whereas 8-Br-cAMP increased promoter activity 2–5-fold (P < 0.05) (Figure 6
). Transient transfection with the 235 bp StAR promoter construct was also undertaken because this promoter exhibits greater (~3-fold) basal activity than the full-length (1.3 kb) promoter (Christenson et al., 1999). Comparison of basal StAR promoter activity (relative luciferase units; mean ± SD) of the 1.3 kb (11830 ± 6375) and 235 bp (36070 ± 421) StAR promoter constructs also indicated a ~3-fold difference in the present experiments. StAR promoter activities for cells transfected with the 235 bp promoter construct and treated with IGF-I (40223 ± 6770), IGF-II (36260 ± 1417) and insulin (40082 ± 1907) failed to exhibit a change in reporter activity compared with controls (36070 ± 421). Similar to the 1.3 kb StAR promoter, cells transfected with the 235 bp and treated with 8-Br-cAMP exhibited increased StAR promoter activity (62951 ± 9249). Collectively, the results of the promoter studies demonstrate that IGF-I, IGF-II and insulin cannot cause a statistically significant increase in the function of a StAR promoter fragment that is responsive to 8-Br-cAMP.
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Discussion
The conversion of cholesterol into pregnenolone is the first step in the biosynthesis of steroid hormones. It is now well accepted that StAR is essential for the translocation of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane where the P450 cholesterol side-chain cleavage enzyme resides (Strauss et al., 1999). Trophic hormones, particularly LH/HCG acting through the cAMP signalling pathway, up-regulate several steps in the steroidogenic process (Miller, 1988), including StAR mRNA and StAR protein expression (Strauss et al., 1999). A number of investigators have shown that in-vitro follicular and luteal steroidogenesis is modulated by factors in addition to gonadotrophin, including intragonadal growth factors (Gougeon, 1996). The present investigation extends our knowledge of regulation of StAR expression in ovarian cells to insulin, IGF-I and IGF-II.
This is the first study to demonstrate an effect of IGF-I, IGF-II or insulin on StAR gene expression (mRNA) and protein values in human granulosa–lutein cells. Previous experiments, with human, rodent and domestic animals' ovarian cells suggested that IGFs and insulin act synergistically with FSH to promote steroidogenesis and follicular growth (see reviews by Adashi et al., 1985; Guidice, 1992). Recently, it was demonstrated that IGF-I causes StAR mRNA accumulation (2.7-fold) in cultured porcine granulosa cells (Balasubramanian et al., 1997). Additionally, these investigators observed that FSH alone and the protein kinase A agonist, 8-Br-cAMP, increased granulosa cell StAR mRNA expression 1.6–3.5-fold, respectively. Interestingly, these investigators observed that combination of FSH or 8-Br-cAMP with IGF-I caused a synergistic increase in StAR expression (26–40-fold). Similarly, these studies on human granulosa–lutein cells showed that IGFs and insulin alone increased StAR mRNA expression 2.5 to 3-fold over control values. 8-Br-cAMP also increased StAR mRNA expression 4-fold. However, in contrast to the observations on pig granulosa cells, we found an additive, not a synergistic increase, in StAR mRNA expression. The different magnitudes of the response between the human granulosa–lutein cells and the porcine granulosa cells could be explained by the different endocrine status of these cells at the time of collection (human peri-ovulatory follicles versus early 1–5 mm antral pig follicles), culture conditions (long-term versus short-term cultures) as well as species differences (Balasubramanian et al., 1997).
Our laboratory previously reported that 8-Br-cAMP stimulates StAR mRNA expression by increasing StAR gene transcription as assessed by nuclear run-on assays (Kiriakidou et al., 1996). Furthermore, through promoter analysis it was determined that basal as well as cAMP responsiveness of the StAR promoter is dependent on three DNA-binding sites for the orphan nuclear receptor, steroidogenic factor-1 (SF-1) (Sugawara et al., 1997). Deletion of these sites blocked the ability of the StAR promoter to respond to 8-Br-cAMP. The results of the present study confirmed that the 1.3 kb and 235 bp StAR promoters are responsive to 8-Br-cAMP. However, the failure to detect in this study an effect of IGF-I on StAR promoter activity in the human proliferating granulosa–lutein cells demonstrates that IGF-I increases StAR mRNA expression by a mechanism that is distinct from cAMP. An IGF/insulin responsive region of the StAR promoter may lie further upstream from this 1.3 kb promoter fragment or in intronic sequences of the gene. Alternatively, IGFs and insulin may increase StAR mRNA expression by post-transcriptional mechanisms. IGF-I alone was not shown to able to increase porcine StAR promoter activity (LaVoie et al., 1999a). However, in contrast to the data presented here, these investigators reported that IGF-I could stimulate FSH/forskolin/8-Br-cAMP-dependent increases in StAR promoter activity. Interestingly, IGF-I only caused an ~25% increase in forskolin and 8-Br-cAMP dependent StAR promoter activity and this was observed at the highest doses of forskolin and 8-Br-cAMP. These small increases in StAR promoter activity do not support the notion of a synergistic interaction between maximal cAMP signalling and IGF-I at the level of transcriptional regulation. Notably, these same authors recently reported that IGF-I treatment increased the stability of low density lipoproteins (LDL) receptor mRNA in porcine granulosa cells (LaVoie et al., 1999b). Similarly, other authors (Zhang et al., 1998) observed that LH receptor gene activity as assessed by transient transfection of the LH-receptor promoter into a mouse Leydig cell line was not enhanced by IGF-I. These authors concluded that IGF-I treatment increased LH receptor mRNA stability. Collectively, these data suggest that the mechanism underlying the additive effect of IGF-I + 8-Br-cAMP treatment on human granulosa–lutein cells could be due to an enhancement of StAR mRNA stability.
IGF-I, IGF-II and insulin are already known to be important regulators of granulosa cell proliferation, differentiation and steroidogenesis (Adashi et al., 1985; Guidice, 1992). In a recent study (Yuan and Guidice, 1999) IGF-II was shown to mediate the effects of FSH on human preantral follicular steroidogenesis (oestrogen synthesis) and growth. Moreover, previous studies by their laboratory and others demonstrated that FSH action in antral follicles was also mediated by IGF-II (Guidice, 1992). Our study confirms the stimulatory effects of IGF-I, IGF-II and insulin on granulosa–lutein cell steroidogenesis. The majority of studies with human granulosa cells utilize IGF-II because IGF-II mRNA is localized to these cells and IGF-I mRNA is absent within the ovarian follicle. However, the human preovulatoy follicle contains significant amounts of IGF-I, suggesting that this growth factor is derived from the vascular system and can have an effect on granulosa cell function. For these reasons we used both IGF-I and IGF-II in our experiments and, in all but two cases, we observed similar responses related to steroidogenesis and StAR mRNA and protein expression. Moreover, the effects of IGF-I and IGF-II are most likely mediated by a single receptor, the type I-IGF receptor, found on the surface of human granulosa–lutein cell (Hernandez et al., 1992). Paradoxically, it was not possible to demonstrate an effect of IGF-I or insulin on progesterone secretion by the proliferating human granulosa–lutein cells, even though amounts of StAR protein were increased. In contrast, 8-Br-cAMP treatment of these cells caused both an increase in StAR and progesterone production. These results suggest that the proliferating human granulosa–lutein cells may have insufficient amounts of an essential component in the steroidogenic pathway (e.g. cholesterol, P450scc, 3ß-hydroxysteroid dehydrogenase, etc) which is not up-regulated by IGF treatment, but is by 8-Br-cAMP. Since these cells were initially grown in medium containing serum (exogenous source of cholesterol) and then transferred to serum-free medium before and during growth factor treatment, these cells may not have had sufficient time to up-regulate 3-hydroxy-3-methylglutryl coenzyme A (HMG-CoA), the rate-limiting enzyme in de-novo cholesterol synthesis, or had sufficient cholesterol stores to support increased steroidogenesis. Treatment with 8-Br-cAMP is known to up-regulate HMG-CoA in human granulosa cells (Golos and Strauss, 1998). Moreover, the progesterone data from the proliferating granulosa–lutein cells contrast with our results with luteinized granulosa cells collected from IVF patients, where IGF, insulin and 8-Br-cAMP stimulated increases in StAR were associated with parallel increases in progesterone secretion.
Human granulosa cells collected at the time of oocyte retrieval and cultured for 24 h displayed the greatest amounts of StAR protein and progesterone production. The abundance of StAR protein and progesterone in human granulosa are not unexpected because of the in-vivo exposure of the cells to HCG. The enhancement of StAR protein and steroidogenesis in luteinized human granulosa cells by 8-Br-cAMP, IGFs and insulin after 6 days in culture indicates that multi-hormonal regulation of StAR protein could play a crucial role in the rescue of the human corpora lutea during the cycle of conception (Devoto et al., 1995).
The effects of insulin on StAR expression and progesterone synthesis are consistent with the recent report (Willis et al., 1998) which found that IGF-I stimulates human periovulatory granulosa cell steroidogenesis to a greater extent than insulin. Importantly, the steroidogenic effect of insulin was observed with low doses of insulin (20 nmol/l) suggesting that the insulin effect on steroidogenesis is mediated through its own receptors and not through the type I IGF receptor.
In summary, the present study provides new insight into the regulation of StAR expression in human granulosa–lutein cells. Our findings document that IGF-I, IGF-II and insulin are able to increase granulosa cell StAR mRNA expression and protein amounts. Furthermore, it was demonstrated that the IGF/insulin-induced increases in StAR expression are not due to an increase in StAR proximal promoter activity, in contrast to 8-Br-cAMP treatment. Thus, IGF/insulin is likely to enhance StAR mRNA expression by a post-transcriptional mechanism such as enhancement of mRNA stability. These IGF/insulin effects on StAR mRNA result ultimately in increased amounts of StAR protein and steroid output by human granulosa–lutein cells.
Acknowledgments
This research was supported by NIH grants HD-06274 and D43-TW-00671. The authors thank Ms Judith Wood for assistance in the preparation of this manuscript.
Notes
3 To whom correspondence should be addressed at: 1354 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104, USA 
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Submitted on May 4, 1999; accepted on August 9, 1999.
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