Mol. Hum. Reprod. Advance Access originally published online on May 21, 2004
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Molecular Human Reproduction, Vol. 10, No. 7, pp. 473-479, 2004
Molecular Human Reproduction vol. 10 no. 7 © European Society of Human Reproduction and Embryology 2004; all rights reserved
Selective alterations in insulin receptor substrates-1, -2 and -4 in theca but not granulosa cells from polycystic ovaries
1Department of Obstetrics and Gynecology, Cedars–Sinai Burns and Allen Research Institute, Cedars–Sinai Medical Center/The David Geffen School of Medicine at UCLA, 8700 Beverly Blvd., Davis 2066, Los Angeles, CA 90048, USA 2Department of Obstetrics and Gynecology, 2nd Clinic of Surgical Gynecology, University School of Medicine, 8 Jaczewski Street, 20-090 Lublin, Poland
3 To whom correspondence should be addressed. Email: magoffin{at}cshs.org
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Abstract |
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The elevated insulin concentrations that occur in many women with polycystic ovary syndrome (PCOS) can contribute significantly to ovarian hyperandrogenism. The objective of the present study was to compare the content of proximal insulin signalling molecules in theca and granulosa cells between polycystic ovaries and regular cycling controls. Individual follicles
were obtained from 11 women with PCOS and 10 regularly cycling control women. The theca and granulosa cells were microdissected from each follicle. Total protein was extracted and signalling proteins were measured by western blot analysis. There was no difference in insulin receptor content between PCOS and controls in either theca or granulosa cells. Insulin receptor substrate (IRS)-1 and -2 were increased 
but IRS-4 was decreased 
in PCOS theca cells. There were no changes in IRS-1, -2 or -4 in granulosa cells. IRS-3 was undetectable in all samples. There were no changes in phosphatidyl inositol-3 kinase catalytic subunits p110
or p110ß in either theca or granulosa cells. These data demonstrate cell-specific alterations in IRS protein concentrations in theca cells from polycystic ovaries that are consistent with an exaggerated amplification of the insulin signal and which may play an important role in ovarian hyperandrogenism and thecal hyperplasia. Key words: granulosa cell/insulin receptor substrate/insulin signalling/PCOS/theca cell
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Polycystic ovary syndrome (PCOS) is a common disorder in pre-menopausal women (Polson et al., 1988
) characterized by chronic anovulation and an accumulation of small antral follicles in the ovary (Goldzieher and Green, 1962). Among the metabolic abnormalities associated with PCOS, hyperandrogenism is a diagnostic feature of the syndrome.
Theca cells are the source of androgen biosynthesis in the human ovary (Erickson et al., 1985). In PCOS the theca interna of many follicles contains significantly more steroidogenic cells than control follicles at a similar developmental stage (Mahajan, 1988) and the theca cells appear to be hyperstimulated. Not only is androgen secretion enhanced in theca cells from polycystic ovaries (Gilling-Smith et al., 1994), the theca cells overexpress mRNA for key genes involved in androgen biosynthesis including LH receptor, StAR, CYP17 and CYP11A (Jakimiuk et al., 2001) and have an increased rate of CYP17 gene transcription (Wickenheisser et al., 2000). Taken together these data indicate that many follicles in polycystic ovaries have an excessive number of theca cells and that these theca cells have increased capacity to synthesize androgens on a per cell basis.
In contrast to the apparent hyperstimulation of theca cells, granulosa cells exhibit characteristics of hypostimulation in PCOS. Proliferation of the granulosa cells is suboptimal as evidenced by the paucity of granulosa cells in the small antral follicles compared to the follicles of regularly cycling controls at a similar developmental stage (Mahajan, 1988). The granulosa cells are unable to fully differentiate in PCOS as demonstrated by their failure to express CYP19. Growth of the follicles is arrested at the stage where the granulosa cells would normally express CYP19 and begin to secrete estrogen (Jakimiuk et al., 1998).
Insulin resistance accompanied by a mild compensatory hyperinsulinaemia is a common feature of women with PCOS (Carmina et al., 1992; Meirow et al., 1995). Although the insulin-resistant women with PCOS maintain normal circulating glucose concentrations, moderately elevated insulin concentrations are required to do so (Legro et al., 1998). There is considerable evidence suggesting a causal link between hyperinsulinaemia and increased ovarian androgen production (Velazquez et al., 1994; Ehrmann et al., 1995; Nestler and Jakubowicz, 1996; Dunaif, 1997). Therefore it is of interest to determine the mechanisms whereby insulin regulates ovarian androgen biosynthesis.
Studies have been conducted regarding the mechanisms of insulin action in women with PCOS using fibroblasts, adipocytes and skeletal muscle cells. Insulin regulation of target cell responses is initiated by insulin binding to a heterotetrameric receptor consisting of two and two ß subunits (Jacobs and Cuatrecasas, 1981) wherein the subunits serve as insulin binding sites and the ß subunits possess tyrosine kinase activity (Roth and Cassell, 1983; Kahn, 1985). Examination of the insulin receptor in women with PCOS revealed no mutations or structural abnormalities (Talbot et al., 1996).
Insulin binding causes autophosphorylation of specific tyrosine residues of the ß-subunit that activates the receptor and initiates transduction of the intracellular signal. A 30% decrease in receptor autophosphorylation after insulin stimulation was found in PCOS adipocytes (Ciaraldi et al., 1992). Glucose transport into the cells following insulin stimulation was comparable to controls but required an 8-fold greater insulin concentration. In contrast to the finding in adipocytes, there were no alterations of insulin sensitivity and responsiveness in PCOS fibroblasts (Ciaraldi et al., 1998) indicating that alterations of insulin action are cell type specific in PCOS.
Activation of the insulin receptor increases phosphorylation of intracellular substrates, principally insulin receptor substrate (IRS) proteins. Four IRS proteins, IRS-1, IRS-2, IRS-3 and IRS-4, have been identified (Sun et al., 1991, 1995; Sciachitano and Taylor, 1997; Lavan et al., 1997a,b). Phosphorylated IRS links activated insulin receptors with mediators of downstream signalling, including phosphatidylinositol-3-kinase (PI3K), Fyn, Grb-2 and Crk (Virkamaki et al., 1999). It was recently shown that IRS-2 levels were increased in muscle cells of women with PCOS, while the levels of IRS-1 were unchanged (Dunaif et al., 2001). The IRS-1-associated PI3K activity was decreased in PCOS skeletal muscle after insulin stimulation (Dunaif et al., 2001), but no difference in IRS-1 associated PI3K activity was detected in PCOS fibroblasts (Book and Dunaif, 1999). These observations provide additional evidence that there are significant differences in insulin signalling among various insulin-responsive tissues.
Although IRS-1 and -2 are major mediators of insulin action, recent data indicate that IRS-3 and -4 have divergent effects on various insulin-stimulated endpoints. In mice lacking IRS-4, there was decreased breeding efficiency and a tendency toward mild insulin resistance (Fantin et al., 2000). In contrast, overexpression of IRS-3 or -4 in mouse embryonic fibroblast cells caused a decrease in IRS-2 protein concentrations. The cellular responses indicated that IRS-3 and -4 may suppress the functions of IRS-1 and -2 (Tsuruzoe et al., 2001). Like IRS-1 but not IRS-2, IRS-4 is strongly associated with Grb-2 and mediates insulin-stimulated MAP kinase activity in 32D myeloid progenitor cells (Uchida et al., 2000). Both IRS-1 and -2 strongly bind p85/ß, which activates PI3K, PKB/Akt and P70s6k, and promotes phosphorylation of BAD. Unlike IRS-1 and -2, IRS-4 only weakly activated p85-associated PI3K activity and did not activate p70s6k. Thus, it appears that while IRS-4 has many features in common with IRS-1 and -2, IRS-4 seems to play a role in insulin signalling distinct from IRS-1 and -2. The role of IRS-3 and -4 have not been examined in PCOS.
Although there is evidence for alterations in the proximal insulin signalling events in PCOS, the tissue-specific nature of these changes dictates that the effects of insulin resistance in PCOS on theca cell androgen production and granulosa cell differentiation can only be determined by directly examining insulin signalling in human theca and granulosa cells. There have not been any published studies regarding proximal insulin signalling in either ovarian cell type. Therefore, the objective of the present study was to compare the content of proximal insulin signalling molecules in theca and granulosa cells from polycystic ovaries with regular cycling controls.
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Materials and methods |
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Subjects
Ovarian tissue specimens were obtained from 11 women with PCOS undergoing electrocauterization of the ovarian surface or wedge resection for the treatment of their infertility and from 10 regularly cycling, age-matched (
years) pre-menopausal control women in the follicular phase of their menstrual cycle undergoing total abdominal hysterectomy and bilateral oophorectomy for uterine leiomyoma or cervical cancer. Women with PCOS were identified based on a history of oligo/amenorrhoea, hirsutism, and typical morphological appearance of polycystic ovaries (normal or enlarged ovarian volume with multiple subcapsular cysts <8 mm in diameter) at laparotomy or laparoscopy with no evidence of hyperprolactinaemia, Cushing's syndrome, congenital or non-classical adrenal hyperplasia, thyroid disease, or hormone-secreting tumours. All subjects had not received hormonal treatment or ovarian suppression for 
3 months prior to obtaining the specimens. Fasting serum samples were obtained prior to surgery and frozen until assayed for total testosterone by radioimmunoassay (DPC, USA). Informed consent was obtained from all subjects participating in the study as approved by the Ethics Committee at the University School of Medicine in Lublin and the institutional review board at Cedars–Sinai Medical Center.
Granulosa and theca cell collection
The ovarian specimens were immediately placed into ice-cold Medium-199 (Gibco BRL, USA) containing 25 mmol/l HEPES and 1 mg/ml bovine serum albumin. After washing off the blood, the ovaries were placed under a dissecting microscope and the follicular fluid was completely aspirated from the follicles using a Hamilton syringe. The follicular fluid volume was measured and the granulosa cells were collected by centrifugation. The follicular fluid was frozen at –80°C until androstenedione and estradiol were measured by radioimmunoassay (DPC). The follicle diameter was calculated from the volume of aspirated fluid. The follicle was opened with microscissors and the granulosa cells were gently scraped from the follicle wall with a platinum loop and collected by flushing with medium. The granulosa cells were centrifuged and the pellet was pooled with the granulosa cells collected from the follicular fluid. The theca interna was microdissected from the follicle wall after the granulosa cells had been removed. The isolated granulosa and theca cells were frozen at –80°C until the proteins were extracted. Twenty-one control follicles 3–7 mm in diameter from regularly cycling women and 37 follicles 3–7 mm in diameter from women with PCOS were analysed.
Western blot analysis
Proteins were extracted from the isolated theca or granulosa cells using Tri-Reagent (MRC, USA) according to the manufacturer's protocol. Protein pellets were solubilized in 1% sodium dodecyl sulphate (SDS) and the concentrations were determined by NanoOrange assay (Molecular Probes, Inc., USA) according to the manufacturer's protocol. Proteins 
from each sample were separated in 4–12% Bis–Tris gradient gels and transferred to PVDF membranes (Hybond-P; Amersham Pharmacia Biotech, Inc., USA). Membranes were blocked for 1 h at room temperature with TBST (10 mmol/l Tris, pH 8.0, 150 mmol/l NaCl and 0.05% Tween-20) containing 5% (wt/vol) non-fat milk.
Blots were incubated with anti-IR (kindly provided by Dr Richard Roth, University of California, San Francisco; 1:500 dilution), anti-IRS-1 (C-20, Santa Cruz Biotechnology, Inc., USA; 1:350 dilution), anti-IRS-2 (H-205, Santa Cruz Biotechnology; 1:300 dilution) or anti-IRS-3 (S-20, Santa Cruz Biotechnology; 1:500 dilution) antibody overnight at 4°C in blocking buffer. The anti-IRS-1 antiserum is a rabbit polyclonal antiserum raised against a carboxy terminal peptide that does not cross-react with other IRS proteins. The anti-IRS-2 antiserum is a rabbit polyclonal antiserum specific for IRS-2 that was raised against an internal peptide. The anti-IRS-3 antiserum is a goat polyclonal antiserum raised against a carboxy terminal peptide of rat origin that does not cross-react with other IRS proteins. Membranes were washed (three times, 10 min each time) with TBST and incubated with donkey anti-mouse (IR), donkey anti-rabbit (IRS-1 and -2) or donkey anti-goat (IRS-3) antibody conjugated to alkaline phosphatase (Chemicon International, Inc., USA; 1:10 000 dilution) for 1 h at room temperature in blocking buffer. The blots were washed (three times, 10 min each time) at room temperature with TBST. Immunoreactive proteins were detected by incubating in 7-hydroxy-9H-(1, 3-dichloro-9, 9-dimethyl-acridin-2-one) (DDAO)-phosphate (1:3000 dilution) in 10 mmol/l Tris, pH 9.5, 1 mmol/l MgCl2 buffer. Blots were scanned and quantified with the Typhoon imaging system (Molecular Dynamics, Inc., USA).
To measure IRS-4, p110 and p110ß proteins, membranes were submerged in methanol to remove the fluorescent precipitates and then stripped of the antibody by incubating in buffer containing 100 mmol/l 2-mercaptoethanol, 2% SDS (w/v), 62.4 mmol/l Tris, pH 6.7 for 30 min at 50°C. After washing, the membrane was incubated with blocking solution and then incubated with IRS-4 (Upstate Biotechnology, USA; 1:1000), p110 (B-10, Santa Cruz Biotechnology; 1:200 dilution) or p110ß (H-198, Santa Cruz Biotechnology; 1:200 dilution) antibody overnight at 4°C in blocking buffer. The anti-IRS-4 antiserum is a rabbit polyclonal antiserum raised against a carboxy terminal peptide that does not cross-react with other IRS proteins. The anti-p110 antibody is a mouse monoclonal antibody raised against a peptide in the amino terminal region that does not cross-react with p110ß, p110
or p110
. The anti-p110ß antibody is a rabbit polyclonal antiserum raised against a peptide in the amino terminal region that does not cross-react with p110, p110 or p110. After a wash step, blots were incubated with biotin–goat anti-mouse (p110) or anti-rabbit (IRS-4 and p110ß) IgG conjugate (Zymed Laboratories Inc., USA; 1:5000 dilution) for 1 h at room temperature in blocking buffer. After another wash step, blots were incubated with alkaline phosphatase–streptavidin conjugate (Zymed Laboratories; 1:1000 dilution) for 1 h at room temperature. Blots were washed again and the subsequent procedures of fluorescence detection were as described above. To control for sample loading, the blots were stripped as described above and probed with a mouse anti-GAPDH monoclonal antibody (Chemicon International, USA; 1:500 dilution). The fluorescence values for the mean of the control group for each protein were arbitrarily set to 1.0. The fluorescence values for each protein were then divided by the fluorescence values for GAPDH.
Statistical analysis
Differences between groups were compared by t-test. The level of significance was considered to be 
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The subjects with PCOS had a higher body mass index
versus 
and total serum testosterone 
versus 
compared to control subjects (
and 
respectively).
To evaluate whether the proximal signalling molecules were altered in ovarian cells from polycystic ovaries, protein extracts of granulosa or theca cells were analysed by western blotting. There was no difference in GAPDH between control and PCOS groups in either theca or granulosa cells (data not shown). We first compared the amount of insulin receptor- (IR) protein between cells isolated from 3–7 mm follicles in control and polycystic ovaries. As shown in Figure 1, there were no significant differences in IR content in either granulosa or theca cells.
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Both theca and granulosa cells express three isoforms of insulin receptor substrate, IRS-1, IRS-2 and IRS-4 (Figure 2
). IRS-3 protein was not detectable in either granulosa or theca cells (Figure 2). There were 
2-fold higher concentrations of both IRS-1 (Figure 3) and IRS-2 (Figure 4) proteins in theca cells from polycystic ovaries compared to controls. In contrast, there were no differences in the amounts of IRS-1 or -2 in granulosa cells. The content of IRS-4 was decreased in theca cells but not granulosa cells from polycystic ovaries (Figure 5). These data demonstrate that there are isoform-specific and cell type-specific alterations of IRS protein concentrations in theca cells from polycystic ovaries.
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PI3K is an important mediator of the insulin augmentation of thecal 17
-hydroxylase activity (Munir et al., 2004). To determine if increased PI3K could play a role in ovarian hyperandrogenism, PI3K catalytic subunit (p110) content was measured. Two isoforms of p110, namely p110 and p110ß, were expressed in both theca and granulosa cells (Figures 6 and 7). There were no significant differences in the content of p110 (Figure 6) or p110ß (Figure 7) proteins in either granulosa or theca cells between control and polycystic ovaries. Thus, it appears that there is not a generalized increase of insulin signalling intermediates in theca cells from polycystic ovaries.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The protein contents of proximal insulin signalling molecules were determined and compared in theca and granulosa cells from polycystic ovaries and in size-matched control follicles from regularly cycling women in the present study. The results demonstrate the presence of IR
, IRS-1, IRS-2, IRS-4 and PI3K catalytic subunits p110 and p110ß in both theca and granulosa cells from control and polycystic ovaries. No IRS-3 protein was detectable in either human theca or granulosa cells. Although IRS-3 is present in rodents (Sesti et al., 2001), a recent attempt to clone the human IRS-3 gene revealed the absence of a functional IRS-3 gene in humans (Bjornholm et al., 2002). Therefore, IRS-3 can be eliminated from consideration as a mediator of insulin action in human cells.
The effects of insulin on thecal androgen biosynthesis in PCOS is mediated by the insulin receptor (Willis and Franks, 1995; Nestler et al., 1998) and the defects of insulin action in PCOS are thought to be post receptor binding events (Dunaif, 1997; Dunaif and Thomas, 2001). Although some studies showed a lower insulin receptor binding affinity in PCOS adipocytes from both lean and obese patients (Marsden et al., 1994, 2001), the majority of studies in adipocytes, fibroblasts and skeletal muscle cells from PCOS demonstrated no change in receptor number and ligand binding affinity (Ciaraldi et al., 1992; Dunaif et al., 1995, 1992, 2001; Book and Dunaif, 1999). In this study, the content of insulin receptor protein was not altered in either granulosa or theca cells from polycystic ovaries, consistent with the previous observations in non-ovarian tissues. Thus, alterations in insulin receptor concentration are not a cause of ovarian hyperandrogenism.
Many important effects of insulin on target cells are mediated through phosphorylation of the IRS proteins. A study using immunohistochemical staining in women with normal ovulation showed that IRS-1 is the primary insulin receptor substrate in all compartments of the follicle, whereas IRS-2 expression was restricted to the theca cells (Wu et al., 2000). The same study reported a decrease in IRS-1 in granulosa cells but an increase in IRS-2 staining in theca cells from polycystic ovaries. Our data demonstrate an increase in both IRS-1 and -2 in theca cells but not granulosa cells from polycystic ovaries. The increase in IRS-2 protein content in theca cells from PCOS is consistent with the previous study. However, our results show no change in IRS-1 protein in granulosa cells and an increase in IRS-1 protein in theca cells. Furthermore, we clearly demonstrated the presence of IRS-2 in both granulosa and theca cells. The differences are likely to be due to differences in tissue processing and the difficulty in quantifying immunohistochemistry.
Studies in skin fibroblasts indicated that some of the post-insulin receptor dysfunction seen in PCOS may be related to alterations in serine and tyrosine phosphorylation of the insulin receptor (Dunaif et al., 1995). Consistent with previous reports, no reduction in insulin binding was detected. However, 50% of the PCOS subjects' insulin receptors demonstrated modestly reduced autophosphorylation and diminished tyrosine kinase activity. Phosphoamino acid analysis revealed significant decreases in the phospho-tyrosine content and increased constitutive insulin receptor serine phosphorylation. Serine phosphorylation of the insulin receptor has been demonstrated to inhibit the receptor's tyrosine kinase activity (White and Yenush, 1998). A perturbation in the tyrosine kinase function of the insulin receptor should impair phosphorylation of IRS molecules. Although a decrease in IRS-1-associated PI3K activity was observed in skeletal muscle of PCOS subjects (Dunaif et al., 1995), there was no defect in PI3K activation in response to physiological insulin concentrations in PCOS fibroblasts. Thus, the increases in IRS-1 and -2 concentration could compensate for diminished insulin receptor tyrosine kinase activity and maintain insulin responsiveness in theca cells of women with PCOS.
Although IRS-1 and -2 share many structural and functional characteristics, they may play distinct physiological roles due to different tissue distribution, differential expression, different kinetics of activation/deactivation, and/or their specificity in recruiting downstream signalling molecules (Sun et al., 1997; Sesti et al., 2001). Studies from knockout mice suggest that IRS-2 mainly mediates metabolic pathways, whereas IRS-1 preferentially mediates mitogenic pathways of insulin signalling (Tamemoto et al., 1994; Withers et al., 1998). If the increases in IRS-1 and -2 concentrations in PCOS theca cells lead to increased concentrations of phosphorylated IRS proteins, these changes could cause excessive amplification of downstream insulin signals. Due to the method used to collect the samples, the phosphorylation state of the IRS proteins could not be reliably determined in the present study. Based on the results of the murine knockout studies, our data are consistent with the hypothesis that IRS-1 and -2 overexpression could contribute to thecal hyperplasia and excess androgen production respectively. Future studies are planned to examine the functional effects of elevated IRS-1 and -2 in theca cells. In contrast, the concentrations of IRS-1 and -2 did not change in granulosa cells from polycystic ovaries. Thus, it is unclear what role, if any, IRS-1 and -2 may play in the granulosa cell phenotype in PCOS.
IRS-4 is expressed in many human tissues including reproductive organs such as the hypothalamus, pituitary and ovary (Giovannone et al., 2000; Uchida et al., 2000). Our results extend the previous findings by demonstrating that IRS-4 was expressed in both theca and granulosa cells. In theca cells from polycystic ovaries there was a decrease in IRS-4 concentration. This result is intriguing in light of studies in which IRS-4 null mice showed decreased breeding efficiency and a tendency toward mild insulin resistance, both symptoms that are reminiscent of PCOS (Fantin et al., 2000). Our results are also consistent with putative amplification of IRS-1 and -2-mediated signals. Because IRS-4 overexpression in cultured cells suppressed the functions of IRS-1 and -2 at several steps (Tsuruzoe et al., 2001), a decline in IRS-4 concentration would be expected to enhance IRS-1 and -2 function. Further studies are in progress to test this hypothesis.
These new data demonstrate that although there are not generalized alterations of the proximal components of the intracellular insulin signalling pathways, there are cell-specific alterations in IRS protein concentrations in theca cells from polycystic ovaries. The increases in both IRS-1 and -2 concentrations are likely to result in increased concentrations of phosphorylated IRS-1 and -2 proteins selectively in theca cells. These changes may augment the amplification of downstream insulin signalling pathways leading to increased thecal proliferation and androgen production. The decrease of IRS-4 in PCOS theca cells argues in favour of unrestrained activation of PI3K associated with tyrosine-phosphorylated IRS-1 and -2. The increases in IRS-1 and -2 coupled with decreased IRS-4 are consistent with an exaggerated cell-specific amplification of the insulin signal in PCOS theca cells that may play an important role in ovarian hyperstimulation. Although the total concentration of PI-3 kinase is not altered in PCOS, the increased content of IRS-1 and -2 in PCOS theca cells may lead to increased PI3K activity. We have recently shown that insulin stimulation of thecal 17-hydroxylase activity is mediated by PI3K (Munir et al., 2004). Thus, increased PI3K activity, interacting with the cAMP/PKA signalling pathway, should lead to increased androgen production. Further studies will be required to evaluate the specific roles of each IRS protein in hyperandrogenism and thecal hyperplasia in PCOS.
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Acknowledgements |
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We thank Dr R.A.Roth (Stanford University, CA, USA) for kindly providing us with insulin receptor-
antibody. This work was supported by NICHD grant HD041610 (D.M.) and University School of Medicine grant 122/01. |
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Submitted on January 21, 2004; resubmitted on April 6, 2004; accepted on April 13, 2004.
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