Profile of Daughters and Sisters of Women With Polycystic Ovary Syndrome: The Role of Proband's Glucose Tolerance.

CONTEXT: First-degree relatives of women with polycystic ovary syndrome (PCOS) present hormonal and metabolic alterations compared to girls unrelated to PCOS. It is unknown whether glucose intolerance in the PCOS proband confers a more severe metabolic predisposition on their first-degree relatives. OBJECTIVE: To determine whether glucose tolerance status in women with PCOS is associated with worsened glucose metabolism and sex hormone levels in their peripubertal daughters or sisters. DESIGN: Cross-sectional study. SETTING: Seven academic centers in North America, South America, and Europe. PATIENTS: Sixty-four pairs of women with PCOS and their daughters or younger sisters aged between 8 and 14 years were recruited. Twenty-five mothers or older sisters with PCOS were glucose intolerant (GI) and 39 were normal glucose tolerant (NGT). MAIN OUTCOME MEASURES: Beta-cell function estimated by the insulin secretion-sensitivity index-2 (ISSI-2) during an oral glucose tolerance test and by the disposition index during a frequently sampled IV glucose tolerance test. Free testosterone and 17-hydroxyprogesterone (17-OHP) levels. RESULTS: Being related to a GI PCOS proband was associated with a lower ISSI-2 (P-value = 0.032) after adjusting for ethnicity, body mass index z-score, and pubertal stage. They also had higher free testosterone (P-value = 0.011) and 17-OHP levels compared to girls with an NGT proband, the latter becoming significant after adjusting for confounders (P-value = 0.040). CONCLUSIONS: Compared to first-degree female relatives of women with PCOS and NGT, first-degree relatives of women with PCOS and GI display lower beta-cell function and hyperandrogenemia, putting them at higher risk of GI and PCOS development.

Adipocytes from women with polycystic ovary syndrome demonstrate altered phosphorylation and activity of glycogen synthase kinase 3.

OBJECTIVE: To test the hypothesis that an abnormality in glycogen synthase kinase-3 (GSK3) is a pathogenic factor in polycystic ovary syndrome (PCOS). DESIGN: Prospective experimental study (adipocytes). SETTING: Tertiary-care academic medical center and teaching hospital. PATIENT(S): Twenty patients with PCOS and 21 healthy control women. INTERVENTION(S): Blood sampling, physical exam, biopsy of SC lower abdominal fat. MAIN OUTCOME MEASURE(S): Glucose transport and protein levels and phosphorylation state of glycogen synthase kinase (GSK)-3alpha and GSK3beta in adipocytes; assessment of GSK3beta activity. RESULT(S): Basal protein levels of glycogen synthase kinase (GSK3alpha and GSK3beta) did not differ between control women and women with PCOS, nor did basal or insulin-stimulated levels of serine phosphorylated GSK3alpha. However, in adipocytes of women with PCOS, insulin stimulation was not associated with increased serine phosphorylation of GSK3beta, in contrast to the case of control women. Tyrosine phosphorylation of GSK3beta also was higher in women with PCOS, compared with in control women. Consistent with the phosphorylation data, GSK3beta activity was elevated in PCOS adipocytes. CONCLUSION(S): These data suggest that GSK3beta is hyperactivated and resistant to down-regulation by insulin in PCOS. By using physiologic approaches, we demonstrated that abnormal GSK3beta regulation is a potential mechanism for the insulin resistance that is seen in some women with PCOS, which may contribute to their development of the syndrome.

Ovarian enzyme activities in women with polycystic ovary syndrome.

Studies using primary ovarian tissue and cultured human theca and granulosa cells have shown that steroidogenic enzyme activities are up-regulated in theca cells in polycystic ovary syndrome (PCOS). Although granulosa cells in arrested follicles in PCOS fail to express significant amounts of aromatase, there is an overexpression of 5alpha-reductase activity and premature expression of cholesterol side-chain cleavage cytochrome P450.

Mutagenesis of putative serine-threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity.

OBJECTIVE: To investigate the role of serine-threonine phosphorylation on the activity of human P450c17. DESIGN: In vitro study. SETTING: Academic basic research laboratory. PATIENT(S): None. INTERVENTION(S): P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. MAIN OUTCOME MEASURE(S): 17alpha-Hydroxylase and C(17-20) lyase activities of human P450c17. RESULT(S): C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17alpha-hydroxylase and C(17-20) lyase activities than the wild type. CONCLUSION(S): Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.

Dietary galactose inhibits GDF-9 mediated follicular development in the rat ovary.

Clinical evidence suggests an association between galactosemia and premature ovarian failure, but the mechanism is still not fully understood. Growth differentiation factor-9 (GDF-9) is thought to be an obligatory growth factor during the gonadotropin-independent phase of folliculogenesis. The objective of this study was to examine the effects of galactose on initiation of folliculogenesis in the peripubertal interval and the connection between galactose toxicity and GDF-9 expression in the ovary. After immature Long-Evans rats (n = 10) were fed a diet consisting of 20% galactose for 19 days, whole body, ovary and uterine weights were measured. Serum estradiol and progesterone concentrations were measured by radioimmunoassay. Ovarian follicles were counted by morphometric analysis and GDF-9 expression was investigated by immunohistochemistry and immunoblot assay. Galactose treatment did not affect the onset of puberty as marked by the time of vaginal opening. The galactose diet significantly decreased the number of healthy growing follicles. The results of immunoblot assay showed that both bands corresponding to propeptide and mature forms of GDF-9 decreased with the galactose diet about 90 and 70%, respectively. The results of immunohistochemical staining showed that the GDF-9 positive follicle number and the ratio of GDF-9 positive to GDF negative (primordial/non-growing) follicles significantly decreased with this high galactose diet. The present study suggests that a high galactose diet inhibits follicular development, possibly through down-regulation of GDF-9 in the rat ovary, implying that GDF-9 may be involved in galactose-related ovarian toxicity.

Resistin stimulation of 17alpha-hydroxylase activity in ovarian theca cells in vitro: relevance to polycystic ovary syndrome.

CONTEXT: A newly discovered hormone resistin has been shown to be increased in women with polycystic ovary syndrome (PCOS). OBJECTIVE: The purpose of this study was to confirm increased resistin concentrations in women with PCOS and to test the direct effect of resistin on human theca cell androgen production. DESIGN: Resistin was measured in fasting serum samples by RIA. To test the direct effects of resistin on ovarian androgen biosynthesis, human theca cells were cultured with resistin for 3 d in the presence and absence of forskolin and insulin. PATIENTS: Fasting serum samples were obtained from 45 women with PCOS and 74 regularly cycling premenopausal control women in the follicular phase of their menstrual cycles, and ovarian theca cell cultures were established from two control women. RESULTS: The mean serum resistin concentration was increased (40%) in women with PCOS. Serum resistin concentrations correlated positively with body mass index and testosterone in PCOS women but not in controls. There were no significant correlations between resistin and fasting insulin or indicators of insulin resistance when corrected for body mass index. In cultured human theca cells, basal 17alpha-hydroxylase activity was unchanged by resistin alone, but resistin enhanced 17alpha-hydroxylase activity in the presence of forskolin or a combination of forskolin plus insulin. Resistin (> or =1 ng/ml) augmented forskolin and forskolin plus insulin stimulation of CYP17 mRNA expression in a concentration-dependent manner. CONCLUSION: These data indicate that abnormal resistin secretion in PCOS may play a role in causing ovarian hyperandrogenism.

Ovarian theca cell.

Theca cells are the endocrine cells associated with ovarian follicles that play an essential role in fertility by producing the androgen substrate required for ovarian estrogen biosynthesis. Theca cells differentiate from the interfollicular stroma in response to proteins secreted from growing follicles. The most common endocrine cause of infertility is associated with excessive proliferation of theca cells and ovarian hyperandrogenism. Cell facts: -ovarian androgen-producing cells; -are associated only with developing follicles; -over-activity of theca cells causes infertility due to hyperandrogenism; -under-activity of theca cells causes infertility due to lack of estrogen. Theca cells: androgen-producing cells in the ovary.

Estrogen receptor alpha and beta expression in uterine leiomyomas from premenopausal women.

OBJECTIVE: To measure messenger RNA levels of estrogen receptor (ER) alpha and beta in uterine leiomyomas, normal myometrium, and endometrium. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Seventeen premenopausal women who underwent surgery due to symptomatic uterine leiomyomas. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative RT-PCR was used for evaluation of ERalpha and ERbeta expression. Results were normalized to total cellular DNA. RESULT(S): Expression of ERalpha and ERbeta did not differ between leiomyomas and myometrium; however, in both tissues, expression of ERalpha was significantly higher than that of ERbeta. Estrogen receptor-alpha expression in endometrium was lower than in leiomyomas and myometrium. In leiomyomas and endometrium, correlations between expression of ERalpha and ERbeta were found. CONCLUSION(S): Uterine leiomyomas, myometrium, and endometrium display distinct patterns of ER expression.

Differential effects of estradiol, raloxifene and tamoxifen on estrogen receptor expression in cultured human skin fibroblasts.

The balance between ER-alpha and ER-beta in fibroblasts may be crucial in the physiological response to ligands. Up- or down-regulation of the ERs in response to different compounds could mediate the reversal of certain age-related changes in skin and connective tissue. The time-dependent effects of 17-beta estradiol, raloxifene and tamoxifen on ER-alpha and ER-beta mRNA expression in the skin fibroblast cultures were performed. Experiments were carried out in primary cultures of human skin fibroblasts obtained from postmenopausal women. The cells were cultured in medium containing: 2 micromol/l estradiol (E2), 4 micromol/l tamoxifen (Tx) or 4 micromol/l raloxifene (Rx) for 7, 24 and 32 h. ER-alpha and ER-beta mRNAs were measured by quantitative assays based on reverse transcription (RT) of the mRNA and polymerase chain reaction (PCR) amplification of the cDNA. We suggest that ER-alpha and ER-beta are co-expressed in human postmenopausal skin fibroblast and documented that the level of mRNA expression of ERs in this tissue is estradiol, raloxifene or tamoxifen regulated as a mechanism to control the action of those ligands on the cell. On the basis of ER mRNA expression levels, fibroblast response to estradiol appears to be modulated by up-regulation of ER-beta rather than ER-alpha. Two of the examined SERMs appear to have different response to modulation of ERs: response of raloxifen is modulated by up-regulation of ER-beta, and no changes in expression of ER-alpha and tamoxifen response seem to be modulated by ER down-regulation in short-term or up-regulation during longer treatment.

Insulin augmentation of 17alpha-hydroxylase activity is mediated by phosphatidyl inositol 3-kinase but not extracellular signal-regulated kinase-1/2 in human ovarian theca cells.

Polycystic ovary syndrome, characterized by hyperandrogenism and chronic anovulation, is frequently associated with insulin resistance. Ample evidence implicates a role for insulin in the genesis of ovarian hyperandrogenism. The objective of this study was to begin to define the intracellular signaling pathway(s) that mediates insulin regulation of 17alpha-hydroxylase activity in human ovarian theca cells. Third-passage theca cells, isolated from the ovaries of regularly cycling premenopausal women, were used. Insulin alone had no effect on 17alpha-hydroxylase activity or CYP17 mRNA expression but required costimulation with forskolin. At the insulin concentration used (10 ng/ml), a neutralizing antibody to the insulin receptor (but not an antibody to the type I IGF receptor) blocked the insulin stimulation of 17alpha-hydroxylase activity, demonstrating that the effects were mediated by the insulin receptor. Insulin stimulated both phosphatidylinositol-3-kinase (PI3-kinase) and extracellular signal-regulated kinase-1/2 (MAPK) pathways. Specific inhibition of MAPK kinase (MEK) with PD98059 or I0126 did not decrease the 17alpha-hydroxylase activity stimulated by forskolin or forskolin plus insulin. In contrast, the PI3-kinase inhibitor LY294002 completely blocked insulin-stimulated 17alpha-hydroxylase activity. Our data demonstrate that insulin stimulates PI3-kinase and extracellular signal-regulated kinase-1/2 activities in human theca cells, but only PI3-kinase mediates the insulin augmentation of forskolin-stimulated 17alpha-hydroxylase activity.

Metabolic effects of dietary lactose in adult female rats.

As an outgrowth of our interest in the potential toxicity of dietary galactose, we investigated the metabolic effects of high lactose diets in Long-Evans female rats. Seventy-five Long-Evans female rats (25-day-old) were randomized to receive one of 3 diets for 7 months: glucose diet (CON); low lactose diet (10.5%, LLD); or a high lactose diet (41.9%, HLD). Necropsy was performed seven months after randomization. HLD animals had significantly lower body weights than controls (P < 0.01). These animals continued to grow, however at a retarded rate compared to the CON group. The HLD group also had significantly lower triglyceride and non-esterified fatty acid levels than the CON group (P < 0.01 and P < 0.05). Serum glucose concentrations were lower in the HLD group compared to CON animals (P < 0.05), while serum insulin levels were lower than both the LLD and CON animals (P < 0.01 and P < 0.05). Leptin exhibited a similar trend. Thyroid studies revealed no difference in TSH between groups. Free T4 was significantly higher in HLD rats compared to LLD and CON rats while free T3 was lower in the HLD group (P < 0.05). This indicates a possible impairment in T4 to T3 conversion. Our data suggests that a long-term high lactose diet is associated with a decrease in insulin and leptin levels, and an increase in the insulin to glucose ratio. However, these changes are seen in the presence of a decreased body mass. A significant effect on thyroid hormone metabolism is also seen, and may be an adaptive mechanism in lactose-fed rats.

Estrogen receptor alpha and beta expression in theca and granulosa cells from women with polycystic ovary syndrome.

A defining characteristic of dominant follicles is high estradiol concentrations. Abnormal expression of estrogen receptors (ERs) could contribute to poor follicular development and ovulatory failure in polycystic ovary syndrome (PCOS). The aim of this study was to determine whether there are differences in ERalpha and ERbeta expression in granulosa cells (GC) and theca cells (TC) from women with PCOS, compared with regularly cycling women. GC and TC were obtained by microdissection from 12 polycystic and 23 normal ovaries. ERalpha and ERbeta mRNA and protein expression were measured by semiquantitative RT-PCR and Western blot, respectively. In control ovaries, both GC and TC ERalpha mRNAs were higher in small antral (SA) than in dominant follicles. ERalpha mRNA was similar in PCOS and size-matched control follicles. In control follicles, ERalpha protein concentrations were higher in GC than in TC. In GC, the ERalpha concentrations were comparable among SA, dominant, and PCOS follicles. In TC, ERalpha concentrations were lower in dominant follicles but were markedly increased in PCOS. In control ovaries, GC and TC expression of ERbeta mRNA was higher in SA, compared with dominant follicles. In PCOS, ERbeta mRNA was intermediate between SA and dominant follicles in both GC and TC. In GC, the ERbeta protein concentrations followed the same pattern as mRNA expression; but in TC ERbeta, protein in PCOS was equivalent to that in dominant follicles. The results of this study demonstrate that there are significant alterations in the expression of ERalpha and ERbeta in PCOS that may be related to abnormal follicular development.

Human cultured skin fibroblasts express estrogen receptor alpha and beta.

Human skin fibroblasts may be the target cells for estrogens. The aim of present study was to confirm the presence of both isoforms of estrogen receptors (ER) in these cells. Experiments were carried out in primary cultures of human skin fibroblasts. ER-alpha and ER-beta mRNAs were measured by quantitative assays based on reverse transcription (RT) of the mRNA and polymerase chain reaction (PCR) amplification of the cDNA. To determine which of the ER isoforms were present and their intracellular locations immunohistochemical staining was performed. MCF-7 culture was a positive control for the immunostaining. The distribution immunostaining of ER-beta protein differed from that of ER-alpha in skin fibroblasts. ER-alpha was detected in both the cytosolic and nuclear compartments of fibroblasts. ER-beta was weakly detectable and was found predominantly in the nuclear compartment. Using the RT-PCR technique mRNA of both ERs was successfully detected in the skin fibroblast cultures with predominantly higher mean level of ER-beta mRNA expression than ER-alpha mRNA. In human culture skin fibroblasts ER-beta co-expresses with ER-alpha. The dominant expression of ER-beta in cultured female skin fibroblasts suggests that ER-beta may play a dominant role in collaboration with ER-alpha in the regulation of estrogen action in skin.

Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not correlate with polymorphisms in the cholesterol side-chain cleavage and 17alpha-hydroxylase/C(17-20) lyase promoters.

OBJECTIVE: To determine whether overexpression of CYP17 or CYP11A messenger (m)RNA in theca cells from polycystic ovaries is related to polymorphic regions in the gene promoters that may increase transcription. DESIGN: Case-control study. SETTING: Research institute. PATIENT(S): Fifty-one women with PCOS and 280 regularly cycling controls underwent genotyping. Thecal cells were obtained from 23 women with PCOS and 51 controls. MAIN OUTCOME MEASURE(S): Ovarian tissue was obtained from women with PCOS undergoing wedge resection for treatment of their infertility and from controls undergoing ovariectomy for indications unrelated to the study. Expression of mRNA in theca cells was measured by using competitive reverse transcriptase polymerase chain reaction. Genotype analysis for polymorphisms in the CYP11A and CYP17 promoters was performed by using polymerase chain reaction. RESULT(S): Although expression of CYP11A and CYP17 mRNA was higher in women with PCOS, no significant dose effects of CYP11A or CYP17 alleles were observed with respect to serum testosterone; follicular fluid androstenedione, estradiol, and androstenedione-to-estradiol ratio; or CYP11A or CYP17 mRNA expression. CONCLUSION(S): Overexpression of CYP17 and CYP11A mRNA in theca cells from polycystic ovaries is explained by polymorphic differences in the gene promoters.