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O. ORTMANN, H. WIESE and G. EMONS

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O Ortmann, W Asmus, K Diedrich, KD Schulz and G Emons

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.

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O. ORTMANN, R. STURM, R. KNUPPEN and G. EMONS

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O. ORTMANN, G. EMONS, R. KNUPPEN and K.J. CATT

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G. EMONS, U. FINGSCHEIDT, O. ORTMANN, R. STURM and R. KNUPPEN

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G Emons, S Weiss, O Ortmann, C Grundker and KD Schulz

OBJECTIVE: More than 80% of human ovarian cancers express LHRH and its receptor. The proliferation of human ovarian cancer cell lines is reduced by both LHRH agonists and antagonists. This study was designed to further clarify the possible biological function of this LHRH system. DESIGN: As LHRH agonists and antagonists uniformly reduce proliferation of human ovarian cancer in a dose-dependent way, the effect of low concentrations of authentic LHRH was studied. In addition, longer periods of treatment (up to 9 days) were analyzed. To assess the physiological role of LHRH produced by ovarian cancer cells it was neutralized by adequate concentrations of a specific LHRH antiserum. METHODS: Human ovarian cancer cells EFO-21 and EFO-27, which express LHRH and its receptor, were incubated for 1-9 days with increasing concentrations (1pmol/l to 10 micromol/l) of authentic LHRH or with concentrations of LHRH antiserum capable of neutralizing at least 1nmol/l LHRH. Proliferation was assessed by counting cells. RESULTS AND CONCLUSIONS: Authentic LHRH reduced time- and dose-dependently proliferation (by maximally mean+/-s.e.m. 32.7 +/- 4.4%, Newman-Keuls, P < 0.001) of both ovarian cancer cell lines. At very low concentrations (1pmol/l) a marginal reduction of proliferation or no effect was observed. A mitogenic effect of authentic LHRH was never detected. Treatment of ovarian cancer cell cultures with antiserum to LHRH significantly increased (up to mean+/-s.e.m. 121.0 +/- 2.8% of controls, Newman-Keuls P <0.001) proliferation of EFO-21 and EFO-27 cells. These findings suggest that LHRH produced by human ovarian cancer cells might act as a negative autocrine regulator of proliferation.

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G. Emons, O. Ortmann, U. Fingscheidt, P. Ball and R. Knuppen

Abstract. Dispersed pituitary cells from adult female rats were preincubated for different time periods (0– 12 h) in the absence or presence of 10−9 moestradiol (E2) or 4-hydroxyoestradiol (4-OHE2). Then the media were changed and the cells incubated for 4 h with either vehicle, or E2, or 4-OHE2 and additionally with different concentrations (10−11– 10−7 m) of gonadotrophin-releasing hormone (GnRH). Treatment of pituitary cells with E2 for 4 h (i.e. no preincubation with E2) significantly decreased the LH-response to GnRH at concentrations ≥ 10−10 m of the decapeptide. During a transition time of approximately 10 h (i.e. in cultures preincubated with E2 or vehicle for 2, 4, 6 or 8 h and then coincubated with E2 or vehicle and GnRH for 4 h) no differences between E2-and vehicle-treated cultures were observed. After 14 and 16 h of E2-treatment (i.e. 10 or 12 h preincubation and 4 h coincubation with GnRH) the LH-responses to GnRH in these cultures were significantly higher than in the respective controls. A nearly identical reaction pattern was observed when 4-OHE2 was used instead of E2.

In a second series of experiments dispersed rat pituitary cells were suspended in a carrier gel and continuously perifused with medium, using small chromatography columns. When these cells were exposed for 4 min to 10−9 m GnRH at 60 or 48 min intervals, they reacted with reproducible pulsatile LH-discharges during at least 6 subsequent stimuli with the decapeptide. When E2 (10−9 m) was added to the perifusion medium, the LH-responses to GnRH were significantly reduced, starting 36 min after the onset of E2-treatment. These data indicate: 1) In the rat, the negative oestrogen effect is at least in part directly mediated at the pituitary level. 2) The sensitizing effect of oestrogens on rat gonadotrophs to GnRH is significant already after 14 to 16 h. 3) E2 and the catecholoestrogen 4-OHE2 have the same effects in this system. 4) The negative E2-effect on GnRH-induced LH-release is significant after only 36 min, a finding bringing up the question of a non-genomic mechanism.

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G. EMONS, O. ORTMANN, U. FINGSCHEIDT, P. BALL and R. KNUPPEN

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JM Weiss, B Krautmacher, S Polack, K Diedrich and O Ortmann

OBJECTIVE: Recently, GnRH antagonists (GnRHants) have been introduced for the prevention of premature LH surges during controlled ovarian hyperstimulation (COH). Here we investigated whether the GnRHants cetrorelix and ganirelix exert effects on the human ovarian IGF system. Since controversy exists on the action of GnRH agonists in the human ovary, we also tested the effect of triptorelin on IGF-II, IGF-binding protein-2 (IGFBP-2) and pregnancy-associated plasma protein-A (PAPP-A) in cultured human granulosa-lutein cells. DESIGN: In vitro cell culture study in a research laboratory of a university hospital. PATIENTS: Cells were obtained from patients treated with different protocols of COH. In addition to gonadotropins they received triptorelin or cetrorelix. Cells were treated with triptorelin, cetrorelix or ganirelix, 1 nmol/l each, for 48 h. IGF-II, IGFBP-2 and PAPP-A were measured by RIA and enzyme immunoassay respectively. RESULTS: GnRHants and triptorelin did not affect IGF-II, IGFBP-2 or PAPP-A. CONCLUSIONS: We conclude that GnRHants do not exert any significant effects on the IGF system of granulosa-lutein cells and therefore their introduction into protocols of COH is unlikely to impair ovarian function.

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YX Xia, JM Weiss, S Polack, K Diedrich and O Ortmann

BACKGROUND: It is well established that ovarian steroids modulate gonadotropin secretion from anterior pituitary cells. It has been speculated that insulin and IGF-I might influence gonadotropin secretion. OBJECTIVE: To investigate the effects of IGF-I and estradiol alone, or combinations of IGF-I with insulin and estradiol on GnRH-stimulated LH release from female rat pituitary cells in serum-supplemented and serum-free culture conditions. METHODS: Pituitary cells were incubated for 24 h or 48 h with a series of increasing concentrations of IGF-I or estradiol and stimulated with 1 nmol/l GnRH for 3 h. To determine the interaction of IGF-I and estradiol on GnRH-stimulated LH secretion, cells were exposed to increasing concentrations of IGF-I and 100 pmol/l estradiol for 24 h. We also investigated the effects of combined treatment with IGF-I and insulin on GnRH-stimulated LH secretion. RESULTS: Our findings indicate that long-term IGF-I treatment (24 h) alone has a significant augmenting effect on GnRH-stimulated LH release in serum-free medium only, with a maximum at low concentrations (10 and 100 pmol/l). Estradiol significantly increased GnRH-induced LH release in a dose-dependent manner. The extent of GnRH-stimulated LH secretion by long-term estradiol treatment (24 h) was significantly greater in serum-supplemented (+42%) medium than in serum-free medium. Estradiol facilitated IGF-I-primed LH responses to GnRH in serum-free medium. In contrast, in serum-supplemented medium, the facilitating potential of estradiol was lower. We also found that, in GnRH-stimulated cells, LH release was augmented by insulin treatment, in contrast to quiescent cells that had been pretreated with 100 pmol/l IGF-I alone and 1 nmol/l insulin alone. CONCLUSIONS: IGF-I and to a lesser extent insulin stimulate GnRH-induced LH secretion from pituitary gonadotrophs. This action is enhanced by estradiol treatment of the cells. However, the well known stimulatory action of estradiol on LH secretion is dependent on the presence of growth factors.