Koji Iida

Gonadotropin-Releasing Hormone Receptor in Gynecologic Tumors Frequent Expression in Adenocarcinoma Histologic Types

Background. Gonadotropin‐releasing hormone (GnRH) analogs have been used in the therapy of the endocrine‐dependent cancers. The authors attempted to determine the frequency with which Gn‐RH receptor (Gn‐RHR) is present in gynecological cancers.

Tight Coupling of Gonadotropin-Releasing Hormone Receptor to Stimulated Phosphoinositide Turnover and Antigonadotropic Action in Granulosa Cells

Gonadotropin-releasing hormone (Gn-RH) stimulates phosphoinositide turnover by binding to its specific receptor and suppresses gonadotropin-dependent maturation and steroidogenesis in granulosa cells. This study was undertaken to determine whether persistent receptor occupancy was necessary for Gn-RH to exert such actions on rat granulosa cells, or whether Gn-RH actions were continued by a first and transient stimulation by Gn-RH, using a competitive antagonist, antide. Gn-RH stimulated [32P]phosphate incorporation into phosphatidylinositol (PtdIns), which could be terminated by displacement of previously bound Gn-RH from its receptor by antide and restarted by reoccupying the receptors with Gn-RH. Antide could prevent Gn-RH-stimulated PtdIns radiolabelling whenever it was added to incubations. An identical effect of antide was observed also in the anti-follicle-stimulating hormone (FSH) action of Gn-RH. FSH markedly stimulated aromatase activity, and Gn-RH caused a time- and dose-dependent inhibition of FSH action. Estrogen production was quenched by Gn-RH and restarted at a time when Gn-RH was removed from its receptor by antide. These two responses associated with the occupancy of Gn-RH receptor provide the evidence in favor of a tight coupling of stimulated PtdIns turnover to suppression of aromatase activation. These data of required continued activation of receptor might exclude the possibility that hypothalamic Gn-RH participated in the control of steroidogenesis in the ovary.

Clinical evaluation of serum sialosyl‐Tn antigen levels in comparison with CA 125 levels in gynecologic cancers

The serum levels of sialosyl‐α2,6GalNAcαl‐0‐serine/threonine (S‐Tn) antigen and CA 125 antigen were measured in 205 patients with gynecologic tumors, including 48 ovarian cancers, 20 endometrial cancers, 29 cervical cancers, 57 benign ovarian tumors, 37 uterine leiomyomas, and 14 adenomyosis. Using a cutoff value of 41 U/ ml for S‐Tn and 35 U/ml for CA 125, positive findings were obtained in ovarian cancers in 31 of 48 (64.6%) patients with S‐Tn antigen, and in 36 of 48 (75%) patients with CA 125. In uterine malignancies, positive findings were obtained in 11 of 49 (22.4%) patients and in 8 of 49 (16.3%) patients with the serum S‐Tn and CA 125 antigens, respectively. In ovarian benign tumors, false‐positive findings with CA 125 were observed in 16 of 57 (28.1%) patients, but with S‐Tn antigen in only 3 of 57 (5.3%) patients (P < 0.03). For the ovarian tumors, excluding patients with recurrent disease, the specificity, positive predictive value, and accuracy of the serum S‐Tn antigen level for detecting cancer exceeded that of the serum CA 125. The combined assay of serum S‐Tn and CA 125 antigens gave positive results in 38 of 48 (79.2%) patients with ovarian cancers; most of the negative findings were obtained in Stage I disease. A significant decreases in serum S‐Tn level was observed after cytoreductive surgery in 14 patients with ovarian cancer (P < 0.01). Four patients with a subsequent recurrence showed a concomitant rise in serum S‐Tn. The cyst fluid and ascitic fluid showed high levels of S‐Tn antigen in patients with ovarian cancer, in contrast to findings in patients with benign ovarian tumors. In conclusion, serum S‐Tn antigen has limited use in diagnosing early stage ovarian cancer and uterine malignancies, but it can detect with accuracy ovarian cancers when used in a combination assay with CA 125 and can monitor the status of disease after therapy.

Gynecologic tumors and symptoms in childhood and adolescence.

Although tumors are rarely seen in girls, they should be considered in differential diagnosis because of the high incidence of potential malignancy in genital tumors compared with adults. Ultrasonography can quickly evaluate the underlying cause of genital symptoms or signs and allow timely intervention, while magnetic resonance imaging accurately measures the extent and location of the vaginal lesion. As a result of modern progress in the chemotherapy regimens, the prompt and precise detection of either benign or malignant tumors can lead to both cure and preservation of fertility with conservative surgery whenever possible. With the widespread use of obstetrical ultrasonography, fetal abdominal tumors are being diagnosed with increasing frequency. Most of these tumors will undergo spontaneous resolution within the first few months of life.

Direct action of gonadotropin-releasing hormone (LH-RH) analogue on ovary: an alternative acting mechanism of buserelin

SummaryThe mechanisms by which gonadotropin-releasing hormone (LH-RH) analogue buserelin exerts direct action on the ovary was investigated. The analogue inhibited the luteinizing hormone (LH)-induced increase of steroidogenesis by rat ovarian granulosa cells in a dose-dependent manner. The LH-RH analogue did not alter binding capacity and affinity of125I-LH to granulosa cells, suggesting the involvement of post-receptor mechanism. Likewise, the analogue caused inositol trisphosphate (IP3) formation as a result of stimulated inositolphospholipid turnover. Half maximal effects of both steroidogenesis suppression and IP3 production occurred at 10 nM buserelin. These findings indicate that the inhibitory action of buserelin on granulosa cell function is mediated by IP3 (or calcium)-dependent mechanisms. Buserelin, in addition to its well-known action at pituitary level, exerts a direct inhibition of ovarian steroidogenesis at gonadal level without changes in gonadotropin receptors.

STIMULATORY EFFECTS OF ESTROGEN ON GONADOTROPIN-RELEASING HORMONE-INDUCED PHOSPHOINOSITIDE TURNOVER IN GRANULOSA CELLS

Gonadotropin-releasing hormone (Gn-RH) stimulates phosphoinositide metabolism in granulosa cells by binding to its specific receptor, and suppresses gonadotropin-induced steroidogenesis. Incubation of immature rat granulosa cells with Gn-RH stimulated time-sequential [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI) in a dose-dependent manner; EC50 was at 10 nM. Concurrent exposure to estradiol-17 beta (E2) (100 nM) and Gn-RH (1 microM) augmented 32P-labeling of PI by 5-fold, while Gn-RH alone induced 3.5-fold increase in PI-labeling. In cells preincubated with E2 for 48 h, Gn-RH provoked a 7-fold [32P]phosphate incorporation into PI, suggesting the induction by E2 of Gn-RH-responsible phosphoinositide turnover. E2 alone provoked a low but significant increase in basal labeling rate of PA and PI. Progesterone failed to mimic the action of E2. Essentially similar results were also obtained in mature rat granulosa cells. These results indicate that E2 augments Gn-RH-stimulated phospholipid turnover in granulosa cells, and suggest that estrogens within the microenvironment of the ovary may exert a local autoregulatory effect on their own production pathway through accelerating Gn-RH action to attenuate steroidogenesis.

A Frame-Shift Mutation of the Androgen Receptor Gene in a Patient with Receptor-Negative Complete Testicular Feminization: Comparison with a Single Base Substitution in a Receptor-Reduced Incomplete Form

Mutations of the androgen receptor that impair the action of androgens result in abnormal male sexual development. We studied the structure of the androgen receptor gene in a patient with the receptor-negative form of complete testicular feminization and another patient with a receptor-reduced form of incomplete testicular feminization. In the subject with complete testicular feminization, the deletion of a single nucleotide occurred at nucleotide number 1893 at exon 2. The subsequent frame-shift mutation changes the sense of codon 622 from cysteine to a translational stop signal. Codon 622 is in exon 3, so the mutation predicts the synthesis of a truncated receptor that lacks the entire androgen-binding domain. Analysis of a subject with incomplete testicular feminization revealed a single substitution (CGT → CAT) at nucleotide 2675 of exon 7, resulting in the conversion of an arginine at amino acid 840 to a histidine. This mutation in the androgen-binding domain may impair, but not remove, the androgen binding to its receptor. These results suggest that the phenotypes in our subjects are due to the mutations, and that single amino acid substitution and premature termination codon can cause variably severe functional abnormalities.

Estriol binding in uterine corpus cancer and in normal uterine tissues.

1. The specific bindings of estriol (E3) and estradiol-17 beta (E2) to their specific receptors were investigated in endometrial carcinoma from 7 patients and normal tissues from their respective organs or from other patients. 2. In both cytosolic and KCl-extracted fractions from them, specific binding sites for E3 and E2 were detected, demonstrating the presence of their separate receptors in human uterus-associated tissues. 3. In certain cases (6 cases) of well-differentiated adenocarcinoma, the ratio of concentration of E3 receptor to that of E2 receptor was almost equal to or higher than in other normal tissues. 4. These findings of unique localization of E3 receptor distribution may offer new insight into identification of endometrial carcinoma more likely to respond to hormonal influence or therapy.

Phospholipase C in human endometrial fibroblasts and its regulation by estrogens

1. Stimulated inositolphospholipid turnover has been proposed as a signal-transducing mechanism in many cell types. It appears to be initiated by stimulation of hydrolysis of inositolphospholipid by a phospholipase C. 2. In human endometrial fibroblasts, estradiol was observed to cause sequential enhancement of [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), indicating an accelerating effect of estradiol on inositolphospholipid turnover. Specific 32P-radioactivity in the gamma-phosphate of ATP was increased in response to estradiol. Estrone or estriol were without any effects. 3. To investigate possible mechanisms by which estradiol activates a phospholipase C enzyme in the fibroblasts, the plasma membrane fraction isolated from the fibroblasts was exposed to estradiol in the presence of guanosine triphosphate (GTP) to detect inositol trisphosphate (IP3) production. The IP3 production was Ca2+ dependent, a dependency not affected by estradiol. 4. However, ATP decreased the Ca2+ concentration required for IP3 production in a dose-dependent manner; adenosine diphosphate (ADP), cytidine triphosphate (CTP) showed no effects. 5. These findings from cell and cell-free systems might suggest that estradiol stimulates a phospholipase C, as a result of enhancement of intracellular ATP synthesis, but not as a result of a direct effect on the enzyme molecule or direct activation of receptor-phospholipase C unit. 6. This may give us new insight into estrogen-stimulated cellular phenomenon through some mechanisms other than that classically associated with the action of estrogen.

Effects of estradiol-17β and estriol on their binding sites in the rabbit uterus

1. Receptors for estradiol-17 beta (E2) and estriol (E3) were detected in the rabbit uterus. 2. Saturation analysis of estrogen binding sites in the cytosol showed that the dissociation constants of E2 and E3 for the high affinity binding sites were 1.8 +/- 0.5 nM and 2.3 +/- 0.3 nM, respectively, when dextran-coated charcoal was used to isolate free and bound ligands. 3. To eliminate non-specific (cross) bindings to their receptors, effects of unlabeled E2 and E3 on [3H]E3 and [3H]E2 bindings was examined. 4. [3H]E2 cytosol binding was observed to be specific for E2 and [3H]E3 cytosol binding was more specific for E3. 5. E2 priming to rabbits increased the binding sites for both E2 and E3, which was also more potent than E3 priming. 6. Moreover, the increase in E2 binding sites was greater than that in E3 binding sites. 7. These findings may suggest that there are separate binding sites for E2 and E3 in rabbit uterus and that synthesis of their binding sites is regulated by E2 but not E3.