Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation

Polycystic ovary syndrome is the most common cause of anovulatory infertility. Anovulation in polycystic ovary syndrome is characterized by the failure of selection of a dominant follicle with arrest of follicle development at the 5-10 mm stage. In an attempt to elucidate the mechanism of anovulation associated with this disorder we have investigated at what follicle size human granulosa cells from normal and polycystic ovaries respond to LH. Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA. In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5-3.9) and P, 1.0 (0.3-2.5) in ovulatory women and E2, 1.4 (0.7-25.4) and P, 1.3 (0.3-7.0) in anovulatory women. Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.     

Regulation of epidermal growth factor receptor by androgens in human endometrial cells in culture

Women with polycystic ovaries (PCO) have a thicker endometrium than women with normal ovaries. This cannot be due to unopposed oestrogen, as it occurs in ovulatory cycles. Androgens may be involved, as these are raised in women with PCO. The effects of steroids are partly mediated by growth factors and their receptors. The aim of this study was to investigate the effect of androgens on epidermal growth factor (EGF) receptor in human endometrium. Endometrium was enzymatically dispersed and glands and stromal cells separated. Cells were incubated in Ham's F10 medium supplemented with 5% charcoal-stripped fetal calf serum and either androgens or vehicle. Specific binding of [125I]-labelled EGF was measured. Testosterone and dihydrotestosterone (DHT) (10 micromol/l) increased EGF receptor concentration (control 100 +/- 9%, testosterone 196 +/- 23% control; control 100 +/- 1%, DHT 244 +/- 6% control) but did not alter receptor affinity. The effect of testosterone was inhibited by the anti-androgen hydroxyflutamide, but not by the antioestrogen ICI182780 nor the aromatase inhibitor 4-hydroxyandrostenedione. EGF receptor levels were increased by androstenediol (control 100 +/- 2%, androstenediol 120 +/- 10% control) but not by androstanediol, dehydroepiandrosterone (DHA), DHA sulphate or androstenedione. Testosterone and DHT increased EGF receptor concentrations in glandular epithelium (control 100 +/- 24%, testosterone 147 +/- 5%, DHT 185 +/- 30% control). These data suggest that androgens may have an effect on the endometrium via an increase in EGF receptor concentrations.     

Fine needle aspiration biopsy of salivary gland, 1985-1995

Cellular insulin resistance in polycystic ovary syndrome (PCOS) has been shown to involve a novel postbinding defect in insulin signal transduction. To find possible mechanisms for this defect, adipocytes were isolated from age- and weight-matched obese normal cycling (NC) and PCOS subjects. Insulin sensitivity for glucose transport stimulation was impaired in PCOS adipocytes (EC50 = 290 +/- 42 pmol/L) compared to that in NC cells (93 +/- 14; P < 0.005). The lipolytic responses to isoproterenol as well as maximal suppression by insulin were similar in NC and PCOS adipocytes. However, PCOS cells were less sensitive to the antilipolytic effect of insulin (EC50 = 115 +/- 33 pmol/L) compared to NC cells (42 +/- 8; P < 0.01). Treatment of adipocytes from NC subjects with the adenosine receptor agonist N6-phenylisopropyl adenosine had no effect on either insulin responsiveness or sensitivity for glucose transport stimulation. However, N6-phenylisopropyl adenosine treatment was able to normalize insulin sensitivity in PCOS cells (EC50 = 285 +/- 47 vs. 70 +/- 15 pmol/L, before and after treatment; P < 0.05). In conclusion, our results suggest that insulin resistance in PCOS, as accessed in the adipocyte, occurs at an early step in insulin signaling that is common for glucose transport and lipolysis. In addition, this insulin resistance involves an impairment of the system by which adenosine acts to modulate insulin signal transduction.     

Local regulation of primate granulosa cell aromatase activity

Granulosa cells produce inhibin and activin, proteins implicated in the local regulation of preovulatory follicular development. To assess interactions among FSH, LH, inhibin and activin on primate granulosa cell aromatase activity, we studied primary granulosa cell cultures from the ovaries of the common marmoset (Callithrix jacchus), a monkey with an ovarian cycle similar in length to the human cycle. The distinctive action of activin was augmentation of gonadotropin-responsive aromatase activity throughout antral follicular development. FSH-stimulated aromatase activity in granulosa cells from immature follicles was augmented many fold by picomolar amounts of activin. In cell cultures from preovulatory follicles, the presence of activin stimulated basal aromatase activity in the absence of gonadotropin, as well as augmenting the action of LH. Thus, locally produced activin has the potential to modulate aromatase activity in developing ovarian follicles. By contrast, inhibin or inhibin alpha-subunit purified from bovine follicular fluid had minimal effects on aromatase activity. The only significant effect was slight suppression of FSH-inducible aromatase activity in granulosa cells from immature follicles at an inhibin concentration of 100 ng/ml. The finding that inhibin has a negligible effect on aromatase activity in granulosa cells from mature follicles suggests that it is unlikely to exert a physiologically significant influence on aromatase activity in vivo. However, evidence from other studies suggests that inhibin might affect aromatization indirectly through acting locally to modulate thecal androgen (aromatase substate) production. Therefore, both inhibin and activin have the potential to contribute at different levels to paracrine and autocrine regulation of follicular oestrogen synthesis.     

High prevalence of polycystic ovaries and associated clinical, endocrine, and metabolic features in women with previous gestational diabetes mellitus

The prevalence of polycystic ovaries, according to ultrasonography, and associated clinical, endocrine, and metabolic features were investigated in women with previous gestational diabetes mellitus (GDM). Thirty-four women with GDM 3-5 yr before the investigation and 36 controls with uncomplicated pregnancies, selected for similar age, parity, and date of delivery, were investigated. The women with previous GDM showed a higher prevalence of polycystic ovaries [14 of 34 (41%) vs. 1 of 36 (3%); P < 0.0001], hirsutism (P < 0.01), irregular menstrual cycles (P < 0.01), and a higher body mass index (BMI; P < 0.001) than the controls. Five women (15%) with previous GDM had developed manifest diabetes (excluded in comparisons of metabolic variables). After dividing the women with previous GDM into subgroups according to ovarian appearance, the 2 subgroups showed similar glucose tolerance and prevalence of diabetes, whereas the women with polycystic ovaries were younger (mean +/- SD, 33.3 +/- 1.4 vs. 38.2 +/- 1.1; P < 0.01), had higher truncal-abdominal/femoral fat ratio according to skin folds (P < 0.05), had higher concentrations of androstenedione (P < 0.01) and testosterone (P < 0.01), and had a higher LH/FSH ratio (P < 0.01), lower levels of GH (P < 0.01), higher levels of triglycerides (P < 0.05) and cholesterol (P < 0.05) in very low density lipoprotein, all independent of age and BMI, and had a higher prevalence of pregnancy-induced hypertension (50% vs. 15%; P < 0.05) during the index pregnancy compared with the women with normal ovaries. The group of women with GDM showed a lower early insulin release after glucose (i.v. glucose tolerance test) for their degree of insulin resistance (euglycemic hyperinsulinemic clamp) compared with controls (P < 0.05). In the two subgroups, insulin sensitivity was lower in the polycystic ovaries group, independent of BMI (P < 0.05), than in the group with normal ovaries. In conclusion, ultrasonographic, clinical and endocrine signs of polycystic ovary syndrome were much increased in women with a history of GDM. Compared with the women with normal ovaries and previous GDM, those with polycystic ovaries formed a distinct subgroup that may be more prone to develop various features of the insulin resistance syndrome. Both groups showed a similarly disturbed balance between beta-cell activity and insulin sensitivity, but in women with polycystic ovaries, insulin resistance may be the dominant component.     

Developmental changes in marmoset granulosa cell responsiveness to insulin-like growth factor-I: interactions with follicle-stimulating hormone and estradiol

The biological actions of insulin-like growth factor-I (IGF-I) on granulosa cell steroidogenesis at defined stages of preovulatory follicular development in the marmoset monkey were examined. Studies were carried out by primary cell culture of granulosa cells derived from small antral (0.5-1.mm diameter) and large preovulatory (2-3.mm diameter) follicles collected during the mid-late follicular phase of the ovarian cycle. IGF-I (0.3-100 ng/ml) had no effect on progesterone accumulation or aromatase activity during 48-h culture of granulosa cells from small follicles. Progesterone accumulation by cells from large follicles was also unaffected by IGF-I over the same time period, although aromatase activity was stimulated in a dose-dependent manner (18-fold increase over basal levels with a maximally stimulatory dose of 30 ng IGF-I/ml). In contrast, granulosa cells from small and large follicles responded to IGF-I in terms of both progesterone accumulation and aromatase activity after longer periods of culture (4 days for progesterone; 6 days for aromatase). Concurrent treatment of granulosa cells from small follicles with estradiol (10(-7) M) enhanced the dose-dependent actions of IGF-I on both indices of steroidogenesis and advanced the time at which IGF-I stimulated activity was first detectable. The effects of estradiol on granulosa cell IGF-I responsiveness were independent of cell number. A synergistic action of IGF-I on FSH-stimulated granulosa cell steroidogenesis was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of P450 aromatase immunoreactivity in human ovary during the menstrual cycle: relationship between the enzyme activity and immunointensity

To understand changes associated with the menstrual cycle in the human ovary, it is very important to examine chronological changes in P450 aromatase (P450arom) enzymatic activity in the normal cycling ovary. Therefore, we initially examined the correlation between intensity of P450arom immunoreactivity and its biochemical enzymatic activity in five estrogen-producing human cancer cell lines (HHUA, Ishikawa, HEC-59, OMC-2, and MCF-7). P450arom immunointensity per cell was evaluated by the CAS 200 computed image analysis system, and its catalytic activity per 10(6) culture cells was analyzed by the tritiated water method. A significant correlation (r = 0.959) was demonstrated between P450arom immunoreactivity and enzymatic activity under optimal conditions of tissue fixation and immunohistochemical procedures. We then investigated P450arom immunointensity in 31 specimens of normal cycling human ovaries to examine chronological changes in P450arom activity per cell throughout the menstrual cycle. In the follicular phase, P450arom was observed in the granulosa cells of one selected antral follicle per case during the mid- to late proliferative period, and its immunointensity per granulosa cell in the follicle was not significantly different between mid- and late proliferative periods, although serum estradiol level was markedly elevated in the late proliferative period. In the luteal phase, both P450arom immunointensity per luteinized granulosa cell in a corpus luteum and serum estradiol level reached a peak in the mid-secretory period. These findings indicate that different factors may influence ovarian P450arom activity during the follicular and luteal phases, i.e., an increased number of granulosa cells in the selected follicle during the follicular phase but changes in P450arom activity per luteinized granulosa cell in the corpus luteum during the luteal phase.     

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.     

Wortmannin inhibits insulin secretion in pancreatic islets and beta-TC3 cells independent of its inhibition of phosphatidylinositol 3-kinase

Glucose is the primary stimulus for insulin secretion by pancreatic beta-cells, and it triggers membrane depolarization and influx of extracellular Ca2+. Cholinergic agonists amplify insulin release by several pathways, including activation of phospholipase C, which hydrolyzes membrane polyphosphoinositides. A novel phospholipid, phosphatidylinositol 3,4,5- trisphosphate [PtdIns(3,4,5)P3], a product of phosphatidylinositol 3-kinase (PI 3-kinase), has recently been found in various cell types. We demonstrate by immunoblotting that PI 3-kinase is present in both cytosolic and membrane fractions of insulin-secreting beta-TC3 cells and in rat islets. The catalytic activity of PI 3-kinase in immunoprecipitates of islets and beta-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (PtdIns) in the presence of [gamma-32P]ATP. Wortmannin, a fungal metabolite, dose dependently inhibited PI 3-kinase activity of both islets and beta-TC3 cells, with an IC50 of 1 nmol/l and a maximally effective concentration of 100 nmol/l, when it was added directly to the kinase assay. However, if intact islets were incubated with wortmannin and PI 3-kinase subsequently was determined in islet immunoprecipitates, approximately 50% inhibition of PI 3-kinase activity (but no inhibition of glucose- and carbachol-stimulated insulin secretion) from intact islets was obtained at wortmannin concentrations of 100 nmol/l. Wortmannin, at higher concentrations (1 and 10 micromol/l), inhibited glucose- and carbachol-induced insulin secretion of Intact rat islets by 58 and 92%, respectively. Wortmannin had no effect on the basal insulin release from rat islets. A similar dose curve of inhibition of glucose- and carbachol-induced insulin secretion by wortmannin was obtained when beta-TC3 cells were used. Cellular metabolism was, not changed by any wortmannin concentrations tested (0.01-10 micromol/l). Both basal cytosolic [Ca2+]i and carbamyl choline-induced increases of [Ca2]i were unaffected by wortmannin in the presence of 2.5 mmol/l Ca2+, while Ca2+ mobilization from intracellular stores was partially decreased by wortmannin. Together, these data suggest that wortmannin at concentrations that inhibit PI 3-kinase does not affect insulin secretion. PI 3-kinase is unlikely to have a major role in insulin secretion induced by glucose and carbachol.     

Local control of ovarian steroidogenesis

A number of putative paracrine factors are now thought to interact with FSH in the control of ovarian steroidogenesis. The relative importance of these factors remains to be determined, but the presence of the insulin-like growth factors and their binding proteins and the mechanism of control of the latter through the local production of proteases suggests a role for this system in folliculogenesis. We have demonstrated over-production of steroid hormones in tissue from women with polycystic ovaries. Theca cells in monolayer culture produced excessive amounts of progesterone and androstenedione and granulosa cell oestradiol production was considerably enhanced in response to FSH. Recent evidence points to a genetic defect in the expression or translation of steroidogenic hormones as a cause of excess androgen production, but the gene or genes involved has not been established. Data from our group suggest that granulosa cells from anovulatory polycystic ovaries are prematurely matured and we hypothesize that this is due to the interaction of the raised circulating insulin levels with LH in these follicles, an interaction that results in arrested follicular growth.     

Evidence for a single gene effect causing polycystic ovaries and male pattern baldness

OBJECTIVE: Polycystic ovary syndrome is one of the most common endocrine disorders but its aetiology remains unknown. It is highly prevalent within families, suggesting a genetic basic for the syndrome, but the mode of inheritance is unclear. The purpose of this study was to determine the mode of inheritance of polycystic ovary syndrome, within the families of affected individuals, by classic segregation analysis. DESIGN: All first degree relatives of affected individuals were screened for the presence or absence of polycystic ovaries in post-menarchal-premenopausal women and early onset male pattern baldness (MPB) in the males. In extended pedigrees, assignment of affected status in post-menopausal women was made by consideration of the clinical history alone. PATIENTS: Fourteen women (probands), presenting with a variety of clinical symptoms, were identified sequentially as having polycystic ovaries (PCO) by ultrasound scan. They were examined in detail to determine their family structure, clinical and endocrine status. Ten families were found to have sufficient members for further study. MEASUREMENTS: All family members had their body mass index calculated, their degree of hirsutism assessed using the Ferriman and Gallwey score and serum levels of gonadotrophins (FSH and LH), testosterone, prolactin and 17 alpha-hydroxyprogesterone measured by radioimmunoassay. A careful reproductive history was taken for each woman and any menstrual disturbance was noted. Obese probands had their glucose and insulin response to a standard 75-g oral glucose tolerance test determined. Each male family member was also assessed for the degree and time of onset of balding. RESULTS: First degree female relatives of affected individuals had a 51% chance of being affected. Early onset male pattern baldness (MPB) was found to be an accurate phenotype for obligate male carriers. Each family showed autosomal dominant inheritance for PCO with greater than 90% penetrance. CONCLUSIONS: We postulate that PCO and male pattern baldness are caused by alleles of the same gene which affect androgen production or action. The different frequencies of PCO and male pattern baldness arise from differing thresholds for phenotypic expression in females and males respectively. The modifying effects of other genes is the most likely explanation of the somewhat variable phenotype.     

Insulin stimulation of lactate accumulation in isolated human granulosa-luteal cells: a comparison between normal and polycystic ovaries

Polycystic ovary syndrome (PCOS) is often associated with hyperinsulinaemia and peripheral insulin resistance. Whether the ovary is resistant to insulin is a matter of controversy. The aim was therefore to study the effect of insulin on lactate accumulation, an indicator of glucose metabolism, in granulosa-luteal cells from women with PCOS and from women with normal ovarian function. The cells were obtained from women undergoing clinical in-vitro fertilization-embryo transfer, either from patients with normal ovarian function and tubal or male infertility, or from women with PCOS, with or without tubal factor. The patients were down-regulated with buserelin and stimulated with urofollitrophin and human chorionic gonadotrophin (HCG). Follicle aspiration was performed under ultrasound guidance. Following oocyte recovery the granulosa-luteal cells were isolated, washed and cultured (2-3 x 10(4) viable cells/well) in serum-free Eagle's minimal essential medium for 48 h. After washing, the cells were then cultured in medium containing HCG (0.1-10 IU/ml) or insulin (0.05-0.5 microg/ml) for 24-48 h. Lactate accumulation in the media and cellular protein were analysed. Basal lactate accumulation did not differ in granulosa-luteal cells obtained from normal or PCOS ovaries, and averaged 46 and 49 nmol/g protein/24 h, respectively. A significant stimulation (40-60%) was obtained by HCG in both groups. Insulin caused a dose-dependent increase in lactate in granulosa-luteal cells obtained from normal ovaries (control: 45.5 +/- 6.3; insulin 0.5 microg/ml: 77 +/- 10 nmol/microg protein). Lactate accumulation in granulosa-luteal cells from PCOS ovaries was not altered in the presence of insulin. These results suggest that granulosa-luteal cell glucose metabolism is resistant to insulin in PCOS.     

The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells

OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.     

Gonadotropin-releasing hormone agonists administration in polycystic ovary syndrome. Effects on bone mass

Women with amenorrhea and polycystic ovaries (PCO) seem to present a relative degree of protection against bone loss caused by hypoestrogenism. We treated 20 patients affected by polycystic ovary syndrome (PCOS) with a gonadotropin-releasing hormone agonist (GnRH-a) for 6 months. After treatment mean bone mineral density (BMD) significantly decreased. We concluded that patients with PCOS have to be considered at risk of developing osteopenia when treated with GnRH-a. The relative protection against osteoporosis, that is present in amenorrheic patients with PCO, might be attributed to the characteristics of amenorrhea in these patients.     

Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad

Oocyte loss, either directly through attrition (germ cell death) or indirectly through follicular atresia (somatic or granulosa cell death), is a fundamental event associated with defining the time of normal or premature reproductive senescence in females. Although apoptosis has been reported to function as the underlying mechanism responsible for death of both germ cells and somatic cells in the ovary, the final molecular steps which commit ovarian cells to death have not been fully elucidated. To examine if death repressor activity of the bcl-2 gene product is important for germ cell survival, we conducted studies using a Bcl-2 loss-of-function (bcl-2 -/-) transgenic mouse model. Histological analyses revealed that ovaries collected from bcl-2 -/- mice possessed numerous aberrantly formed primordial follicle-like structures containing a single layer of granulosa cells without an oocyte. Additionally, the total number of primordial follicles present which contained a healthy oocyte was markedly reduced in bcl-2 -/- mice as compared to heterozygote (bcl-2 -/+) or wild-type (bcl-2 +/+) mice, suggesting that expression of the bcl-2 death repressor gene is critical for endowment of a normal complement of germ cells and primordial follicles in the mammalian ovary.     

Multiple metachronous metastasis of adenocarcinoma of the kidneys in crossed ectopy with fusion

OBJECTIVE: Hyperandrogenism in patients with polycystic ovary syndrome has been shown to correlate with hyperinsulinaemia of insulin resistance. We have investigated if basal levels of insulin and the response to the intravenous administration of glucagon can reveal insulin resistance in patients with polycystic ovary syndrome. PATIENTS: Nine obese (BMI > 25 kg/m2) and nine non-obese (BMI 19-25 kg/m2) women with PCOS, chosen from a population of 91 women attending the infertility clinic, and 19 normally cycling women (seven obese, 12 non-obese) were studied. Oligo or amenorrhoea, hirsutism, and 12 or more follicles in a given ovary were selection criteria. MEASUREMENTS: Glucagon, 1 mg, was given intravenously to 18 of the 91 women and to the control subjects. Blood was taken at -5, 0, 5, 10 and 15 minutes for measurements of integrated areas under the response curve for insulin, C-peptide and glucose, respectively. Basal blood samples were drawn for fasting insulin, C-peptide, glucose, testosterone, sex hormone-binding globulin (SHBG), free fatty acids and IGF-I measurements. The free androgen index was calculated according to the formula FAI = testosterone x 100/SHBG. RESULTS: There were no significant differences in maximal increment and area under the response curve for glucose, C-peptide and insulin. FAI was significantly higher in all patients with features of polycystic ovary syndrome. However, fasting insulin levels were significantly higher only in obese patients when compared with obese control subjects and lean patients. CONCLUSIONS: The administration of 1 mg glucagon i.v. did not distinguish patients with polycystic ovary syndrome from control subjects. The mild insulin resistance of polycystic ovary syndrome is related only to obesity and is therefore unlikely to play an important role in the hyperandrogenism associated with the syndrome.     

Selective inhibition of primary acute myeloid leukaemia cell growth by lovastatin

The insulin receptors from erythrocytes of 50 patients with non-insulin-dependent diabetes mellitus were tested for their ability to autophosphorylate. The assay was performed by a new enzyme-linked immunosorbent assay system that used monoclonal anti-insulin receptor antibodies absorbed to microtiter plates as a first antibody and polyclonal antiphosphotyrosine antibody as a labeled second antibody. By this assay, 3 patients were identified with defects in their insulin receptor kinase, although their defects appeared heterogeneous. Patient 1 had 85% less maximal autophosphorylation with a normal ED50 (1.6 x 10(-9) M insulin). Patient 2, who had polycystic ovary disease, had a 49.2% decrease in maximal autophosphorylation of insulin receptors, and the ED50 was shifted to the right (5.6 x 10(-8) M). Patient 3 with acanthosis nigricans had a normal maximal autophosphorylation, but the ED50 shifted to the right (2.9 x 10(-8) M). The mechanisms for the diversity detected in this assay is not known, but this technique has sufficient specificity and sensitivity to be used to screen for insulin-resistant patients who have a lack of kinase activity.     

Differential expression of estrogen receptor-beta and estrogen receptor-alpha in the rat ovary

Immunohistochemical localization of two estrogen receptor (ER) subtypes, ER beta and ER alpha, was performed in neonatal, early postnatal, immature, and adult rats to determine whether ER alpha and ER beta are differentially expressed in the ovary. ER beta and ER alpha were visualized using a polyclonal anti-ER beta antibody and a monoclonal ER alpha (ID5) antibody, respectively. Postfixed frozen sections and antigen-retrieved paraffin sections of the ovary revealed nuclear ER beta immunoreactivity (IR) in granulosa cells, which was prevented when peptide-adsorbed antibody was used instead. In immature and adult rat ovaries, ER beta was expressed exclusively in nuclei of granulosa cells of primary, secondary, and mature follicles. Atretic follicle granulosa cells showed only weak or no staining. No specific nuclear ER beta IR was detected in thecal cells, luteal cells, interstitial cells, germinal epithelium, or oocytes. In neonatal rat ovary, no ER beta expression was found. In ovaries of 5- and 10-day-old rats, weak ER beta IR was observed in granulosa cells of primary and secondary follicles, but no staining was detected in the primordial follicles. ER alpha protein exhibited a differential distribution in the ovary with no detectable expression in the granulosa cells but evidence of ER alpha IR in germinal epithelium, interstitial cells, and thecal cells. In the oviduct and uterus, IR for ER alpha, but not ER beta, was found in luminal epithelium, stromal cells, muscle cells, and gland cells. Our present study demonstrates that ER beta and ER alpha proteins are expressed in distinctly different cell types in the ovary. The exclusive presence of ER beta in granulosa cells implies that this specific new subtype of ER beta mediates some effects of estrogen action in the regulation of growth and maturation of ovarian follicles.     

Feedback inhibition of insulin secretion and insulin resistance in polycystic ovarian syndrome with and without obesity

Hyperinsulinemia/insulin resistance is a well-known feature of polycystic ovarian (PCO) syndrome. In this study, the comparative roles of the peripheral tissues and the pancreatic beta-cells in its pathogenesis were evaluated. We determined basal serum C-peptide values (index of insulin secretion) and in vivo insulin action on peripheral glucose utilization (by the euglycemic hyperinsulinemic clamp technique) in obese (n = 5) and nonobese (n = 5) PCO women compared to obese (n = 5) and nonobese (n = 5) normal ovulatory women. During the clamp, feed-back inhibition of insulin on insulin secretion was studied by C-peptide percentage suppression. Serum C-peptide basal values did not differ significantly between the four groups. Insulin stimulated glucose utilization, expressed as M-value, was significantly decreased in both PCO groups compared to normal ovulatory women (p < 0.005). The metabolic clearance rate of glucose (MCR) and insulin (M/I) had the same behaviour. No differences were found between M, MCR and M/I values and the two groups of PCO subjects (obese/nonobese). The C-peptide percentage suppression was similar in all the groups. We conclude that PCO women have a significant insulin resistance that is independent of obesity, while basal and insulin-inhibited insulin secretion do not differ from normal-cycle subjects.     

An aminopeptidase inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine granulosa cells stimulated with follicle stimulating hormone in vitro

We examined the presence of cell surface aminopeptidase on cultured porcine granulosa cells by employing the aminopeptidase assay using alanine-p-nitroanilide and histochemical staining using L-leucyl-beta-naphthylamide. Porcine granulosa cells obtained from follicles 4-5 mm in diameter were cultured for 7 days. The aminopeptidase assay showed that the porcine granulosa cell culture had aminopeptidase activity and that this activity was inhibited in a dose-dependent manner by bestatin which binds to cell surfaces and inhibits cell surface aminopeptidases. Histochemical staining also indicated that cultured granulosa cells had aminopeptidase activity. Porcine granulosa cells were cultured in the presence or absence of porcine follicle stimulating hormone (FSH, 3.125 nmol/l) and/or bestatin (0.4, 4.0 and 40.0 micrograms/ml) for 7 days, and the production of progesterone and oestradiol was measured. In the presence of porcine FSH, the production of progesterone and oestradiol by granulosa cells was increased significantly by approximately 5- and 2-fold respectively. These increases were enhanced further by bestatin (40.0 micrograms/ml). In the absence of porcine FSH, progesterone production was enhanced by bestatin (40.0 micrograms/ml), whereas no significant effect of bestatin on oestradiol secretion was observed. These findings indicate that the inhibition of membrane-bound aminopeptidase(s) on the cell surfaces affects the steroidogenesis of granulosa cells, and that these aminopeptidase(s) are important regulators of granulosa cell differentiation.