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The aim of this study was to explore the direct action of noradrenaline and dopamine on progesterone and estradiol secretion of human granulosa cells cultured in serum-free medium. Progesterone and estradiol production was measured in the presence and absence of noradrenaline, dopamine or propranolol using radioimmunoassays; statistical analysis was performed by analysis of variance and Newman-Keul's multiple range test. Twenty-six women aged 31 +/- 3 years undergoing in vitro fertilization and embryo transfer for infertility treatment at University Women's Hospital, University of Tübingen, Germany, took part in this study. Noradrenaline significantly inhibited progesterone production by human granulosa cells in a dose-related manner at a concentration of 10(-4)-10(-6) mol/l. Dopamine significantly stimulated estradiol secretion by granulosa cells in an inverse dose-related manner. Both effects were blocked by propranolol. The results suggest that catecholaminergic actions switch over the steroid production of human granulosa cells cultured in serum-free medium from progesterone to estradiol.

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Cholinergic effects on hormone secretion by human granulosa cells (GCs) are not well characterized. The aim of this study was to explore the direct action of acetylcholine and carbachol on progesterone and estradiol secretion of human GCs cultured in serum-free medium. Granulosa cells were obtained from 26 women undergoing in vitro fertilization and embryo transfer. Progesterone and estradiol production was measured in the presence and absence of acetylcholine, carbachol, or atropine using radioimmunoassays; statistical analysis of the data was performed by ANOVA. Acetylcholine significantly stimulated progesterone secretion by GCs in a dose-related manner. Estradiol secretion was also stimulated by acetylcholine, but this effect did not show dose dependency. Carbachol showed a similar stimulatory effect, but to a lower degree; both effects can be blocked by acetylcholine. The results suggest that cholinergic action on steroid production by human GCs is mediated through the muscarinic route, and cholinergic neurotransmission may have a physiological significance in the intra-ovarian regulatory pathways.

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Noradrenaline (NA), serotonin (5HT), dopamine (DA), FSH, LH and prolactin (PRL) content was determined in 104 preovulatory follicular fluids obtained from 44 patients undergoing in vitro fertilization. The patients were given human menopausal gonadotropin (HMG) for ovarium stimulation, ovulation was induced with 10000 IU human chorionic gonadotropin (HCG) 34-36 hours prior to the follicular aspiration by vaginal ultrasound. Classification of the oocytes was performed by direct microscopic evaluation differentiating three groups of oocytes: Group I.: prophase I; Group II.: metaphase I; Group III.: metaphase II. There was no significant difference in monoamine and FSH content of follicular fluid at different stage of the oocyte maturation. LH and PRL significantly increased parallel with oocyte maturation (38.9; 48.8; 56.7 IU/l and 1324; 2382; 3134 IU/l). significant negative correlation was observed in Group I. between 5HT-LH (r = -0.64); in Group II. between NA-LH (r = -0.62) and NA-PRL (r = -0.51). Significant positive correlation were found in Group I. between FSH-LH (r = 0.63), in Group II. between LH-PRL (r = 0.56), in Group III. between NA-5HT (r = 0.66), NA-DA (r = 0.80) and 5HT-DA (r = 0.66). These observations suggest that action of LH and PRL may be negatively modulated by 5HT and NA in the final stage of oocyte maturation.

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OBJECTIVE: To explore the direct action of serotonin on progesterone (P) and estradiol (E2) secretion of human granulosa cells cultured in serum-free medium. DESIGN: Progesterone and E2 production was measured in the presence and absence of serotonin, propranolol, or cycloheximide using radioimmunoassays; statistical analysis of the data was performed by ANOVA. SETTING: In vitro fertilization and embryo transfer (IVF-ET) for infertility treatment at the University Women's Hospital, University of Tübingen, Germany. PATIENTS, PARTICIPANTS: Fourteen women, 30 +/- 3 years old, undergoing IVF-ET. RESULTS: Serotonin stimulated a dose-related increase in P secretion with a maximal stimulatory effect at 10(-4) M. This response was blocked specifically by the beta-receptor antagonist propranolol (10(-4) M). Estradiol secretion in response to serotonin was dose-independent stimulation, which was highest at 10(-6) M and was inhibited by 10(-4) M propranolol. The protein synthesis inhibitor cycloheximide markedly reduced the stimulatory effect of serotonin on P secretion. Estradiol production in the presence of cycloheximide was significantly reduced; serotonin had no stimulatory effect under these conditions. CONCLUSION: Serotonin may have a physiological role in the corpus hemorrhagicum, when luteinization is initiated.

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Anovulatory patients with clomiphene-resistant polycystic ovarian syndrome were treated by two different stimulation protocols. Follicular maturation was induced in 14 women with hMG; 12 of them received pure FSH in a later series after previous pituitary desensitization with the LHRH agonist D-Trp-6-LHRH (Decapeptyl). Both basal and stimulated serum androstenedione, testosterone, and free testosterone were elevated in the hMG-treated group compared with controls. However, only androstenedione exhibited a significant increase between early and late follicular levels. Marked suppression of these androgens has been observed after two weeks of LHRH agonist pretreatment, but nearly the same concentrations were obtained with pure FSH on the day of hCG administration. Again, only the increase of androstenedione levels proved to be significant. Polycystic follicular maturation, hyperstimulation, and peak estradiol levels were comparable with the two protocols. Nevertheless, more pregnancies were achieved using the LHRH agonist+FSH combination (4 vs. 1). It is suggested that 2 weeks' suppression of ovarian androgens with LHRH agonist is not sufficient to neutralize the unfavourable intraovarian mechanisms interfering with normal folliculogenesis. More data are required to confirm the superiority of LHRH agonist pretreatment in the management of polycystic ovarian syndrome.

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Progesterone secretion of cultured human granulosa cells treated with LHRH or its analogues was measured. Granulosa cells were also stimulated with LH, FSH, cAMP or forskolin. No differences could be observed in the progesterone basal secretion of cultures treated with or without LHRH or its analogues and after the various stimulations. It was concluded that the clinical experience that the sensitivity of ovaries for gonadotropins after LHRH desensitization is decreased could not be explained with the peripheral effects of LHRH on the granulosa cells. From the data obtained, the lack of LHRH receptors in the ovaries are suggested in humans.

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Infertile patients who responded poorly in an in-vitro fertilization programme were treated with human menopausal gonadotrophin (HMG) or with pure follicle stimulating hormone (FSH) during continuous administration of a luteinizing hormone-releasing hormone (LHRH) agonist, to determine whether a low level of LH is required for follicle maturation. No statistically significant differences were detected in the dose of gonadotrophins, duration of treatment, oestradiol and LH levels, numbers of recovered oocytes, transferred embryos or fertilization rates. It is concluded that an absence of low levels of LH does not disturb follicular development in the follicular phase. Based on the low fertilization rates in the present study (0.32 with HMG versus 0.45 with FSH) the authors suggest that, as well as hormonal deficiency, other factors may also influence follicular and early embryonic development.

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Granulosa cells from human preovulatory follicles were cultured under serum-free conditions to investigate the presence of immunoreactive insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 levels were measured by a radioimmunoassay developed against the acid-stable subunit of the protein. The antiserum had no cross-reactivity to the low molecular weight GH-independent IGFBP-1. Granulosa luteal cells exhibited a continuous release of IGFBP-3 into the culture medium during the whole time (6 days) of the incubation. A dose-dependent increase in IGFBP-3 was observed when the cells were treated by dibutyryl cAMP. Cycloheximide suppressed almost completely both the basal and the stimulated production of IGFBP-3. The smallest effective dose of dibutyryl cAMP enhancing the progesterone release was lower than that for IGFBP-3. The different time course of IGFBP-3 and progesterone secretion to dibutyryl cAMP treatment, as well as the failure of progesterone to elicit IGFBP-3 increase alone, do not support the participation of progesterone in the IGFBP-3 production of granulosa cells. It is concluded that 1. immunoreactive IGFBP-3 is produced by cultured granulosa luteal cells; 2. its synthesis is regulated by physiological intracellular mechanisms.

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Increasing evidence suggests that insulin-like growth factors I and II (IGF-I, IGF-II) have a regulatory role in animal granulosa cells. This study was undertaken to investigate the presence of IGF-I and IGF-II, as well as that of their binding proteins (BP), IGFBP-1 and IGFBP-3 in human serum and follicular fluid (FF). Preovulatory FF was obtained from 51 patients undergoing in-vitro fertilization. The IGFBP-1 level was found to be significantly higher (P less than 0.01) in FF than in serum, whereas IGF-I and IGFBP-3 values remained markedly lower (P less than 0.01) in FF. Serum IGF-II levels were slightly but not significantly elevated compared to values obtained in the FF of patients. A positive correlation (P less than 0.001) between individual serum and FF levels was observed only for IGF-I. When a group of poor responders was compared to patients with normal stimulation characteristics, no significant difference was found in either IGF or IGFBP levels in the FF. It is concluded that IGFBP-1 is produced locally, whereas the serum may possibly be the major source of IGF-I. No clear conclusions can be drawn regarding the source of FF IGF-II and IGFBP-3. Neither the absolute level nor the relationship of IGFs to their transport proteins could explain the poor response to ovarian stimulation.

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In the absence of hormone stimulation, prophase-blocked oocytes of Marthasterias glacialis have been induced to undergo meiosis reinitiation up to female pronucleus formation by pulse incubation in isoosmotic urea-sea water solutions. Even when this procedure was not effective all along the breeding season, it could trigger full maturation when applied to so-called "incompetent oocytes" that did not complete maturation following microinjection-induced mixing of their nucleoplasm and cytoplasm. 32 P phosphate incorporation into proteins and cell fusion experiments demonstrate that this treatment produces an increased protein phosphorylation which appears tightly associated with the production of M-phase promoting factor (MPF). Instead, when oocytes are maintained in the inducing medium, dephosphorylation soon occurs and MPF is no longer present to support meiosis. Under these conditions, the GV-disrupted oocytes present a permanent nucleolus and do not form a meiotic spindle. The same cytological aspect was also obtained when the oocytes were treated in the presence of 90 µM emetine or 150 µM of the intracellular chelator Quin 2-AM. These data suggest that urea-induced maturation may involve an intracellular Ca2+ shift which would be required to activate both MPF precursor molecules and the resting female centers which stand in the animal cortex outside the nucleus and give rise to the poles of the first maturation spindle. They also show that nuclear disruption alone, without protein phosphorylation, cannot trigger meiosis reinitiation of incompetent oocytes.