Acupuncture regulates the autophagy of ovarian granulosa cells in polycystic ovarian syndrome ovulation disorder by inhibiting the PI3K/AKT/mTOR pathway through LncMEG3.

The main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulation dysfunction, which are caused by the excessive autophagy of ovarian granulosa cells. Acupuncture has been shown to improve ovulation dysfunction and abnormal follicular development in PCOS patients, but its mechanism is unclear. This study hypothesized that the beneficial effects of acupuncture are the result of LncMEG3-mediated effects on the PI3K/AKT/mTOR pathway. Acupuncture (CV-4, RN-3, CV-6, SP-6 and EX-CA 1) was used to treat a rat model of polycystic ovary syndrome. Hematoxylin-eosin staining was used to observe ovarian morphology and enzyme-linked immunosorbent assay, western blotting, immunohistochemistry and real-time PCR were used to detect LH, E2, FSH, T, AMH, LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression. The ovarian morphology of 90% of the rats in the acupuncture treatment group was significantly improved after 11 consecutive days of therapy. Acupuncture also resulted in a significant decrease in serum LH, FSH, T and AMH levels and a significant increase in E2 level (P<0.01). LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression was decreased in ovarian granulosa cells after acupuncture compared with PCOS and lentiviral Intervention Group (P<0.05), while the expression of follicle stimulating hormone receptor was increased (P<0.05). These results indicate that acupuncture can down-regulate the expression of LncMEG3 and thereby inhibit the PI3K/AKT/mTOR pathway, reducing granulosa cell autophagy and normalizing their proliferation. These factors ultimately remedy abnormal follicular development. These findings suggest that acupuncture has clinical potential as a safe treatment for PCOS ovulatory dysfunction.

Upregulation of Cyp19a1 and PPAR-γ in ovarian steroidogenic pathway by Ficus religiosa: A potential cure for polycystic ovary syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Quite a few plants are in use to treat female infertility and associated problems. Availing the cues from traditional knowledge, phytochemical studies and ethnopharmacological evidences, the aphrodisiac plant Ficus religiosa (F. religiosa) is widely in use to cure infertility in women. For instance, the juice of leaf and aerial root of F. religiosa is reported to normalize the dysregulated menstrual cycle in women. Besides, it is believed that regular circumambulation of F. religiosa during the early hours of the morning helps women in alleviating infertility which could be attributed to the potential phytovolatiles released from F. religiosa. However, the evidences for therapeutic potential of F. religiosa in treating female infertility are arbitrary and mostly anecdotal. AIM OF THE STUDY: The present study was aimed at examining if extracts of fresh and/or dry leaf of F. religiosa would cure polycystic ovary syndrome (PCOS) in the rat model. METHODS: Rats were divided into seven groups; control (Group I), PCOS-induced (P.O, Letrozole -1 mg/kg BW for 21 days) and untreated (Group II), PCOS-induced and treated with the leaf extracts of F. religiosa (Groups III-VI), and, PCOS-induced and treated with pioglitazone (Group VII). The estrous intervals, body and organ weights (ovary and uterus), and serum hormones (testosterone, luteinizing hormone [LH], estrogen, and progesterone) were measured, and the expression of Cyp19a1 (aromatase), and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) were assessed in the experimental rats. The levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and antioxidants (MDA, GSH, GPx, SOD, and CAT) were also quantified. Besides, the putative volatile compounds in the esterified leaf extracts were identified using Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: Letrozole treatment induced irregular estrous and altered weight of organs and hormonal milieu, which were reverted to normal in leaf extracts-treated PCOS-induced rats. Remarkably, fresh leaf treatment up-regulated Cyp19a1and PPAR-γ and increased the levels of 3ß-HSD and 17ß-HSD. We found 3-acetoxy-3-hydroxy-propionic acid in fresh and dry leaf extracts, which we attribute to efficacy of the extracts in alleviating PCOS. CONCLUSION: Put together, our findings suggest the leaves of F. religiosa as potential in alleviating PCOS, mainly due to the presence of putative volatile molecules. Further screening of the leaves of F. religiosa is recommended to identify other key molecules and to develop a systematic therapeutic intervention for PCOS.

In Search of New Therapeutics-Molecular Aspects of the PCOS Pathophysiology: Genetics, Hormones, Metabolism and Beyond.

In a healthy female reproductive system, a subtle hormonal and metabolic dance leads to repetitive cyclic changes in the ovaries and uterus, which make an effective ovulation and potential implantation of an embryo possible. However, that is not so in the case of polycystic ovary syndrome (PCOS), in which case the central mechanism responsible for entraining hormonal and metabolic rhythms during the menstrual cycle is notably disrupted. In this review we provide a detailed description of the possible scenario of PCOS pathogenesis. We begin from the analysis of how a set of genetic disorders related to PCOS leads to particular malfunctions at a molecular level (e.g., increased enzyme activities of cytochrome P450 (CYP) type 17A1 (17α-hydroxylase), 3ß-HSD type II and CYP type 11A1 (side-chain cleavage enzyme) in theca cells, or changes in the expression of aquaporins in granulosa cells) and discuss further cellular- and tissue-level consequences (e.g., anovulation, elevated levels of the advanced glycation end products in ovaries), which in turn lead to the observed subsequent systemic symptoms. Since gene-editing therapy is currently out of reach, herein special emphasis is placed on discussing what kinds of drug targets and which potentially active substances seem promising for an effective medication, acting on the primary causes of PCOS on a molecular level.

Does myo-inositol oxygenase, the only enzyme to catalyze myo-inositol in vivo, play a role in the etiology of polycystic ovarian syndrome?

In polycystic ovary syndrome (PCOS), myo-inositol (MI) supplements have shown many beneficial effects. In this study, therefore, we aimed to investigate the serum level of myo-inositol oxygenase (MIOX), which is the only enzyme catalyzing MI in vivo, in patients with PCOS. Serum MIOX enzyme levels and other laboratory parameters were compared between sixty patients, who were diagnosed with PCOS for the first time, and sixty healthy individuals at similar age and sex. MIOX serum levels were not different between two groups (p = 0.7428). MIOX median and 95% CI were 19.4 and 10.6-39.1 in the control group and 16.4 and 7.6-46.2 in the patient group respectively. Demographic data, biochemical and hematological parameters, hormone parameters were not different except from the lymphocyte count between the two groups. Lymphocyte count was higher in the patient group. Although the ratio of LH/FSH was higher in the patient group, it was not statistically significant. Our results suggest that serum MIOX levels do not change in PCOS. It was, therefore, concluded that MI deficiency observed in PCOS was not related to the level of MIOX enzyme which cleaves MI.

Phosphatidylinositide 3-kinase inhibition: A new potential target for the treatment of polycystic ovarian syndrome.

CONTEXT: Quercetin, a flavonoid, has been tried in traditional medicine for treating many disorders and reported to have inhibitory action on PI3 kinase. OBJECTIVE: This study investigates the effect of quercetin on testosterone propionate induced polycystic ovary syndrome (PCOS) model, which shows both metabolic and endocrine features of PCOS. MATERIALS AND METHODS: Female pre-pubertal Sprague-Dawley rats were randomly divided into four groups: normal control, PCOS control, quercetin, and metformin treated. PCOS was induced by testosterone propionate (10 mg/kg, s.c.) and treatments were carried out orally at the dose of 150 mg/kg from the 6th week. At the 6th and 10th week, blood was collected to investigate metabolic indices, and reproductive biochemical parameters including morphology of ovary, uterus, and estrous cyclicity were assessed. The ovaries were processed to determine CYP17A1 gene expression. RESULTS: The treatment with quercetin did not modify body weight gain but uterine (296.7 ± 5.11 versus 263.0 ± 8.60 mg) and ovary weights (49.5 ± 1.93 versus 37.8 ± 3.43 mg) were found to be decreased significantly (p <0.05) as compared with the PCOS control group. The PCOS control group showed hyperinsulinemia, hyperandrogenemia, and dyslipidemia. Treatment with quercetin showed statistically significant (p <0.01) improvement in insulin (12.46 ± 0.3 versus 10.0 ± 0.28 µU/ml), testosterone (0.65 ± 0.02 versus 0.29 ± 0.02 µU/ml), luteinising hormone (20.6 ± 0.28 versus 15.1 ± 0.36 U/ml), and lipid profile. Histological examination of ovary and uterus confirmed the disease occurrence and remission state in the diseased and treated groups, respectively. Quercetin also demonstrated PI3 kinase inhibition in a docking study and decreased CYP17A1 gene expression. DISCUSSION AND CONCLUSION: Thus, we can conclude that quercetin may have beneficial effect in PCOS by virtue of inhibition of PI3K which attributes to a decrease in the expression of CYP17A1 gene, having a key role in steroidogenesis.

Impact of androgen and dietary advanced glycation end products on female rat liver.

BACKGROUND/AIMS: Advanced glycation end products (AGEs) have been related to a wide range of liver disorders including hyperandrogenic states such as the Polycystic Ovary Syndrome (PCOS). The aim of the present study is to evaluate the potential impact of dietary glycotoxins exposure and androgen excess on hepatic histology and biochemistry in an androgenized female rat model. METHODS: The study population consisted of 80 female Wistar rats, divided in 3 groups, a group of prepubertal (Group A, n=30) and adult rats (Group B, n=20) that were androgenized via subcutaneous implantation of dihydrotestosterone-containing pellets as well as a group of adult non-androgenized rodents (Group C, n=30). All groups were randomly assigned either to a high-AGE or low-AGE diet for 3 months. RESULTS: Rats fed with a high-AGE diet exhibited significantly elevated levels of gamma-glutamyl transferase (x03B3;GT) (p≤0.0002) and indices of AGE immunostaining in liver tissue (p<0.01) when compared to the respective low-AGE group, while aspartate aminotransferase (AST) levels were affected only in non-androgenized animals (p=0.0002). Androgenization per se constitutes an aggravating factor as demonstrated by the elevated x03B3;GT levels in adult androgenized animals compared to non-androgenized, independent of diet content (p=0.0002) and by the elevated AST and alanine aminotransferase (ALT) levels in low-AGE subgroups (adult androgenized vs. non-androgenized, p=0.0002) followed by increased immunohistochemical AGE deposition in hepatocytes of the latter categories (p=0.0007). CONCLUSION: The present study suggests that androgens and glycotoxins may contribute synergistically to distort hepatic physiology and function as observed in hyperandrogenic conditions.

[Effects of Lycii Cortex on express of PI3K/PKB in PCOS rats].

The effect of Lycii Cortex on the PCOS rat model and the mechanism of action were investigated in the present study. The PCOS rat model was induced with Poretsky methods. Then the rats were randomly divided into four groups: the model group, melbine group (0.45 g x kg(-1)), low (2.5 g x kg(-1) and high (10 g x kg(-1)) dosage group of Lycii Cortex. The animals were orally administrated with the drugs for 14 days. In addition, another control group was added in this study. The rats were weighted before and after drug treatment. After 14 days treatment, oestrous cycle of rats were detected; blood serum was separated to determine T and FINS and rat's uteri were isolated. The mRNA and protein (total and phosphorylated) expressions of PI3K and PKB in uteri were measured with Real-time RT-PCR and Western blot, respectively. Compared with the control rats, the body weight gain and serum level of T and FINS were significantly increased. While, the mRNA and protein (phosphorylated) levels of PI3K and PKB were markedly decreased in PCOS group. Lycii Cortex treatment significantly decreased the body weight gain and serum level of T and FINS in a dose-dependant manner. It also markedly increased the mRNA and protein (phosphorylated) expressions of PI3K and PKB. Meanwhile, the melbine treatment also showed the curative effect. Lycii Cortex can relieve the symptoms of PCOS and the mechanism might be related to PI3K/PKB pathway.

[Association of methylenetetrahydrofolate reductase gene polymorphisms with polycystic ovary syndrome].

OBJECTIVE: To assess the association of C677T and A1298C polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene with the susceptibility to polycystic ovary syndrome (PCOS). METHODS: Blood samples of 115 PCOS patients and 58 fertile women (for whom PCOS has been excluded) were collected for DNA extraction. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for determining the C677T and A1298C polymorphisms. A database has been set up with Epidata and a significance test was performed with a statistical analysis system. RESULTS: A significant difference has been found in the allele frequencies of MTHFR gene 677 C and T polymorphisms between the two groups (P<0.01), for which T allele has increased the risk for PCOS by 2.06 times. Heterozygous and homozygous genotypes at position 677 (CT and TT) were more common among PCOS cases than controls, with an OR of 3.91 (95%CI: 1.70-8.97) and 4.39 (95%CI: 1.77-10.89), respectively. There was no statistical difference in genotypic distribution of MTHFR gene A1298C polymorphism (P>0.05). In the PCOS group, there was a significant difference with an OR of 6.40 (95%CI: 1.71-23.95) for an increased risk of insulin resistance in homozygous C677T mutations (TT) compared with the wild genotype (CC, P<0.01). The PCOS group and the control group also differed significantly in their red blood cell folate levels (P<0.01), but not in serum vitamin B12 and homocysteine levels (P>0.05). CONCLUSION: MTHFR gene C677T polymorphism is associated with PCOS, for which CT and TT genotypes can increase the risk of PCOS. The TT genotype can also increase the risk of insulin resistance in PCOS patients. The A1298C polymorphism of the MTHFR gene is not associated with the occurrence of PCOS. The folate level in red blood cells of PCOS patients is lower, for whom folate should be supplemented.

Spleen-yang-deficiency patients with polycystic ovary syndrome have higher levels of visfatin.

OBJECTIVE: To study serum visfatin levels in women with polycystic ovary syndrome (PCOS) grouped by Traditional Chinese Medicine (TCM) patterns. To study the correlations of serum visfatin levels with homeostatic model assessment insulin resistance (HOMA-IR), fasting plasma glucose (FPG), fasting insulin (FINS), body mass index (BMI), testosterone (T), total cholesterol (TC), and triglycerides (TG). METHODS: Two hundred and twelve PCOS patients were placed into the following TCM pattern subgroups: Kidney-Yang deficiency (KYD) group, Spleen-Yang deficiency (SYD) group, stagnant Liver-Qi transforming into heat (SLQTH) group, and Kidney-Yin deficiency (KYIND) group. The correlations between serum visfatin levels and HOMA-IR, FPG, FINS, BMI, T, TC, and TG were analyzed. RESULTS: Of all patients with PCOS, there were 82 in the KYD group (38.6%), 67 in the SYD group (31.6%), 37 in the SLQTH group (17.5%), and 26 in the KYIND group (12.3%). Visfatin levels in all PCOS subgroups were higher than those in the control group (P < 0.01 or P < 0.05). Among these subgroups, the visfatin levels in the SYD group were significantly higher than those in the other three TCM pattern groups (P < 0.05). There were no statistical differences among the remaining three pattern groups. The levels of BMI, FINS, HOMA-IR, T, and TG were significantly higher in all subgroups than those in the control group (P < 0.05). There were no significant differences in FPG and TC between all PCOS subgroups and the control group (P > 0.05). The SYD group had higher levels of FINS and HOMA-IR compared with the KYD, SLQTH, and KYIND groups (P < 0.05). In all subgroups, after controlling for BMI, TG, TC, and age, visfatin was positively correlated with FINS (r = 0.197, P = 0.015) and HOMA-IR (r = 0.173, P = 0.033), and was not correlated with T. CONCLUSION: KYD and SYD patterns are most common in PCOS patients. Increased visfatin is a common pathophysiologic manifestation in PCOS patients. The SYD group had the highest levels of visfatin, and visfatin was positively correlated with FINS and HOMA-IR.

[Effects of electroacupuncture of "Guanyuan" (CV 4)-"Zhongji" (CV 3) on ovarian P450 arom and P450c 17alpha expression and relevant sex hormone levels in rats with polycystic ovary syndrome].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on ovarian P 450 arom and P 450 c 17 alpha (aromatases) expression and related sex hormone levels in polycystic ovary syndrome (PCOS) rats. METHODS: Thirty SD rats were randomly divided into normal control group, model group and EA group (10 rats/group). PCOS model was made by intragastric administration of letrozole at 1 mg/kg per day for consecutive 21 days. "Guanyuan" (CV 4) and "Zhongji" (CV 3) acupoints were stimulated 20 min by EA (2 mA, 2 Hz), once daily for consecutive 14 days. The damp ovarian weight was weighed and the pathological changes of the ovarian tissue were observed after H. E. staining. Ultrastructural changes of the ovarian tissue were observed by transmission electron microscope. Immunohistochemical staining was adopted to detect ovarian follicle granulosa cell P 450 arom and follicle membrane cell P 450 c 17 alpha expression. The contents of estradiol (E 2), estrone (E 1), androstenedione (ASD), testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the ovarian tissue were measured by ELISA. RESULTS: Compared with the normal group, there was a significant increase in the damp weight of both left and right ovarian tissues in the model group (P < 0.01). After EA, the ovarian weight was remarkably reduced (P < 0.01). Pathological changes of the ovarian tissue such as thickening of the superficial albugineous coat of the ovary, thinning of the granular cell layer, and disappearance of the intraovular oocytes and coronaradiata under light microscope, and mitochondrion swelling, fracture or disappearance of mitochondrial cristae, and enlargement of the endoplasmic reticulum, etc. after modeling were obviously improved in the EA group. In comparison to the control group, the expression of the follicle granulosa cell P450 arom was significantly down-regulated and that of follicle membrane cell P 450 c 17 alpha was significantly upregulated in the model group (P < 0.01). After EA intervention these changes were obviously reversed (P < 0.05, P < 0.01). In the model group, there was a significant increase in the levels of ASD, T and LH in the ovarian tissues (P < 0.01) and a marked decrease in the contents of ovarian E 1 and E2 (P < 0.01) in comparison to the control group. After EA, the ovarian ASD, T and LH levels were notably decreased (P < 0.05, P < 0.01) and the E 1 and E 2 levels apparently increased (P < 0.01) compared with the model group. CONCLUSION: EA can improve letrozole-induced pathological changes of ovarian morphology and ultrastructure and obviously promote P 450 arom expression in the follicle granulosa cell layer and inhibit P 450 c 17 alpha expression in the follicle membrane cell layer, as well as regulate sex hormone levels in PCOS rats, facilitating the normal transformation of ovarian androgen to estrogen and restoring the local endocrine disorders.

[Effects of bushen tongmai recipe on expression of phosphatidylinositol-3-kinase in PCOS rats accompanying with insulin resistance].

OBJECTIVE: To observe the effects of bushen tongmai recipe (BSTMR) on mRNA and protein expressions of phosphatidylinositol-3-kinase (PI-3K) p85alpha in hepatic, adipose, muscular and ovarian tissues in PCOS rats with insulin resistance (IR). METHOD: Twenty-three-day-old female SD rats were injected subcutaneously with sodium prasterone sulfate (90 mg x kg(-1) x d(-1)) for 20 days, and fed with high-fat forage for 80 days to induce PCOS rats with IR Then the rats were randomly divided into the model group and the treated group. Meanwhile, a group of fifteen rats of the same age was considered as the normal control group. The treated group were administered with BSTMR. The ovulation condition was examined by hematoxylin-eosin (HE) staining. Fasting blood glucose (FBG) was determined using glucose oxidase method. Serum fasting insulin (Fins) was determined by radioimmunoassay (RIA). The mRNA level of PI-3K p85alpha was measured by reverse transcription polymerase chain reaction(RT-PCR). Immunohistochemistry staining was wtilised to detect protein expression of PI-3K p85alpha in ovary. RESULT: Compared with the model group, the mean number of corpus luteum and the rate of ovulation in the treated group increased significantly (P <0. 01). The level of Fins in the treated group was much lower than that in the model group (P < 0.01). Both mRNA and protein expressions of PI-3K p85alpha in target tissues were up-regulated significantly in the treated group (P < 0.01 or P < 0.05). CONCLUSION: BSTMR could improve IR and ovulation dysfunction in PCOS rats accompanying with IR and its molecular mechanisms might be closely related with the elevation of mRNA and protein levels of PI-3K p85alpha in target tissues of the model rats.

Effects of Bushen Tongmai Recipe on protein kinase Balpha expression in polycystic ovary rats with insulin resistance.

OBJECTIVE: To observe the effects of Bushen Tongmai Recipe (, BSTMR) on mRNA and protein expressions of protein kinase B alpha (PKB alpha) in hepatic, adipose, muscular, and ovarian tissues of polycystic ovary (PCO) rats with insulin resistance (IR) and to explore the possible molecular mechanism of BSTMR in treating IR and ovulation dysfunction. METHODS: Female 22-day-old SD rats were injected subcutaneously with sodium prasterone sulfate (9 mg.100g(-1).d(-1)) for 20 days and fed with high-fat diet for 80 days to induce PCO rats with IR. Then, the PCO rats were randomly divided into the model group (n=23) and the treated group (n=21). The treated group was administered with BSTMR for 2 weeks. Meanwhile, a group with 15 rats of the same age was used as the control group. The histological changes in the ovaries were examined. Fasting blood glucose (FBG) was determined by the glucose oxidase method. Serum fasting insulin (Fins) was determined by radioimmunoassay (RIA). The mRNA level of PKBalpha was measured by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry staining and Western blot analysis were employed to detect the protein expression in target tissues. RESULTS: Compared with the control group, the ovaries in the model group showed multiple follicular cysts, levels of FBG and Fins in the model group increased markedly (P<0.05 or P<0.01, respectively), and the insulin sensitive index (ISI) decreased obviously (P<0.01). The mRNA and protein expressions of PKBalpha in target tissues in the model group were dramatically lower than those in the control group (P<0.05 or P<0.01). Compared with the model group, the stratum granulosum of the ovarian follicle in the treated group increased markedly, the level of Fins in the treated group decreased obviously (P<0.01), ISI in the treated group improved markedly (P<0.01), and the mRNA and protein expressions of PKBalpha in target tissues of the treated rats were elevated significantly (P<0.05 or P<0.01). CONCLUSION: BSTMR could improve IR and ovulation dysfunction in PCO rats with IR, and its molecular mechanisms might be closely related with the elevation of mRNA and protein expressions of PKBalpha in target tissues of PCO rats with IR.

Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5-10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17alpha-hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzyme's 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor beta-chain (IR-beta) inhibits IR-beta tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-beta. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-beta serine phosphorylation, and three PCOS patients with increased levels of IR-beta serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17alpha-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17alpha-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells' donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 micromol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-beta, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.