Naoki Terakawa

Changes of bioactive luteinizing hormone after laparoscopic ovarian cautery in patients with polycystic ovarian syndrome

The serum levels of bioactive luteinizing hormone (LH), immunoreactive LH, follicle-stimulating hormone, androstenedione (A), and testosterone (T) were determined in nine anovulatory women with polycystic ovarian syndrome (PCOS) before and after laparoscopic ovarian cautery. Eight ovulated spontaneously and three conceived after treatment. Before treatment, the mean (+/- SEM) levels of bioactive LH, immunoreactive LH, A, and T were 51.4 +/- 8.6 mIU/mL, 36.0 +/- 4.5 mIU/mL, 1.98 +/- 0.35 ng/mL, and 1.18 +/- 0.13 ng/mL, respectively, which were significantly higher than those of five control women (19.2 +/- 1.6 mIU/mL, 21.4 +/- 1.2 mIU/mL, 0.54 +/- 0.03 ng/mL, 0.28 +/- 0.03 ng/mL). After treatment, the mean levels of these hormones had significantly decreased. Decreases in the levels of these hormones by laparoscopic ovarian cautery in women with PCOS may result in both restoration of the ovulatory cycle and achievement of pregnancy.

Hormonal regulation of activities of 17β-ol-dehydrogenases, aromatase and 4-ene-5α-reductase in immature rat ovaries

Abstract Female rats were hypophysectomized at 21 days of age, and after 3 days, the hypophysectomized rats in groups of 3–20 were injected daily with 10 μg of NIH-LH-S19, 10–100 μg of NIAMD-Rat-FSH-B-1, 20 μg of oestradiol-17β or saline for 3 days. Ovarian homogenates from these rats and intact rats at 27 days of age were incubated with [14C]-4-androstene-3,17-dione, [14C]-oestrone, [7-3H]-4-androstene-3,17-dione or [7-3H]-progesterone and enzyme activities and metabolism of progesterone were estimated. The activities of 5α-reductase, testosterone and oestradiol 17β-ol-dehydrogenases and aromatase decreased significantly 6 days following hypophysectomy. A distinct response to LH but not to FSH in the 5α-reductase activity in the hypophysectomized rat ovary was found. On the other hand, the activities of 17β-ol-dehydrogenases and aromatase in the hypophysectomized rat ovary were stimulated (10 to 200 times) by FSH but not by LH. No stimulation of these enzyme activities by oestradiol- 17β was involved. The formation of oestradiol-17β from progesterone could be demonstrated only in the FSH-injected rat ovary. These results show that the 5α-reductase activity is regulated by LH and the activities of 17β-oldehydrogenases and aromatase are regulated by FSH in immature rat ovaries. It is also suggested that nonresponse to LH and response to FSH of the uterus of hypophysectomized immature rats can be explained in part by the present results.

RU486 a progestin antagonist binds to progesterone receptors in a human endometrial cancer cell line and reverses the growth inhibition by progestins.

The human endometrial cancer cell line, IK-90 cells, contains estrogen-independent progesterone receptors (PR) and is progestin sensitive. Accumulation of glycogen in the cytoplasm of IK-90 cells as well as growth inhibition of the cells in response to progestins are observed. In the present study, the effects of RU486, a progestin antagonist, on IK-90 cells were investigated in a serum-supplemented medium. Scatchard plot analysis of cytoplasmic binding data in the cells revealed a high affinity binding site for RU486 (Kd, 2.6 nM) with maximum binding sites of 169 fmol/mg protein. However, the binding ability to DNA-cellulose of heat activated [3H]RU486-PR complexes was lower when compared with that of the progestin agonist [3H]R5020-PR complexes, suggesting a decrease in progestin activity of RU486 in IK-90 cells. The addition of 1 microM RU486 to culture medium produced periodic acid-Schiff-positive granules in the cytoplasm of the cells. On the other hand, RU486 (1 nM-1 microM) did not significantly inhibit the growth of cells. However, RU486 (0.1-1 microM) totally prevented the growth-inhibitory effect of R5020 (0.1-1 microM) on IK-90 cells. In conclusion, RU486, an antiprogestin, had a dual activity both a progestin antagonist and weak agonist in human endometrial cancer cells, which was not mediated through the estrogen receptor system.

Danazol binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro

Based on our recent findings that danazol, an isoxazol derivative of ethinyltestosterone, has a profound growth-inhibitory effect on an established human endometrial adenocarcinoma cell line, the effects of danazol on cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in the present study. Of the 22 uterine adenocarcinomas, estrogen, progesterone, and androgen receptors were found in 12, 14, and 4 tumors, respectively. Competitive binding studies showed that danazol specifically binds to progesterone and androgen receptors but not to estrogen receptors. Of the five cancer cells from five patients succeeded in primary cell culture, a marked inhibition of cell growth was demonstrated by addition of danazol in two cancer cells having progesterone but not androgen receptors. However, danazol did not affect the growth of the remaining three cancer cells lacking progesterone receptors. These results strongly suggest that danazol has a significant growth-inhibitory effect on human endometrial adenocarcinoma cells, possibly through progesterone receptors in the cells.

Preliminary Report on the Use of Danazol in the Treatment of Endometrial Adenomatous Hyperplasia

Since the authors recently reported that danazol inhibits the growth of human endometrial cancer cells in vitro, a clinical trial was initiated to examine whether danazol can regress adenomatous hyperplasia, a precursor of endometrial adenocarcinoma. Danazol was used for 3 months in the treatment of five patients with a history of hypermenorrhea and irregular uterine bleeding. Within a 9‐month follow‐up period, all patients were symptom‐free, and none of the specimens obtained by endometrial biopsy showed the presence of adenomatous hyperplasia. These findings suggest that danazol has a potential application in the treatment of patients with endometrial adenomatous hyperplasia. A possible mechanism of action of the compound in adenomatous hyperplasia is also discussed.

Hormonal regulation of 4-ENE-5α-reductase activity in prepubertal rat ovaries

Abstract Female rats were hypophysectomized at 21 days of age, and after a lapse of 3 days the hypophysectomized rats in groups of 4–13 were injected daily with NIH-LH-S19, NIAMD-Rat-FSH-B-I, 5α-androstane-3α, 17β-diol, testosterone, oestradiol-17β, saline or sesame oil for 3 days. Ovarian homogenates from these rats at 27 days of age were incubated with [ 14 C]-testosterone and 5α-reductase activities were estimated. The 5α-reductase activity (nmol/g tissue/h or nmol/both ovaries/h) decreased significantly 6 days following hypophysectomy. A distinct response to LH in 5α-reductase activity of the hypophysectomized rat ovaries was found, with a dose-response in activity from 260 ± 94 (S.D.) in the hypophysectomized control to the maximum level of 1960 ± 177 (S.D.) nmol/g/h, using doses from 3 to 90 μg per day. In contrast, FSH was not effective, for it had little effect on 5α-reductase activity even when 10 or 50 μg was given each day. No stimulation of 5α-reductase activity by injection of androgens and oestradiol-17β in large doses was involved, showing that the effect of LH is not mediated by sex steroids. These results show that 5α-reductase activity in prepubertal rat ovaries is regulated by LH.

Growth inhibition by danazol in a human endometrial cancer cell line with estrogen-independent progesterone receptors

Since we recently found that danazol, an isoxazol derivative of ethinyltestosterone, has a growth-inhibitory effect on human endometrial cancer cells in primary culture, the effects of danazol on a human endometrial cancer cell line (IK-90 cells), which contains estrogen-independent progesterone receptors (PR), were investigated in the present study. The addition of danazol (1 nM-1 microM) in culture medium caused a decrease in the growth of IK-90 cells in a dose-dependent manner. Competitive binding studies showed that danazol effectively binds to PR in IK-90 cells, and the binding affinity for PR was estimated to be 6.0% of that of R5020. The addition of 1 microM danazol in culture medium resulted in a rapid and significant increase in nuclear PR with a concomitant decrease in cytoplasmic PR in the cells. These findings suggest that danazol has a growth-inhibitory effect on human endometrial adenocarcinoma cells directly through PR system in the cells.

Second pregnancy with spontaneous ovulation following clomiphene- or gonadotropin-induced pregnancy.

For investigation of the rates of spontaneous pregnancy following termination of pregnancy induced by treatment with clomiphene or human menopausal gonadotropin-human chorionic gonadotropin (hMG-hCG) and of spontaneous abortion in the second pregnancy, 119 women (58 with an anovulatory cycle and 141 with amenorrhea) who desired another pregnancy were studied. The rate of spontaneous pregnancy following pregnancy induced by clomiphene was 46.6%, which was significantly (p less than 0.001) higher than that following pregnancy induced by hMG (14.9%). The mean (+/- SEM) period between the termination of the first induced-ovulation pregnancy and the second spontaneous pregnancy was 16.6 +/- 1.4 months. The spontaneous abortion rates in the second pregnancies after pregnancies induced by clomiphene and hMG were 7.5% and 9.8%, respectively, which were significantly (p less than 0.001, p less than 0.05) lower than those in the first pregnancy (25.7%, 33.0%). There was no difference between the spontaneous abortion rates following a first pregnancy which terminated in abortion and those following a pregnancy which ended in birth. These data suggest that a first pregnancy in anovulatory women may restore the ovulatory cycle and have a beneficial effect on the rates of spontaneous abortion in the second pregnancy.

4-Hydroxytamoxifen binds to estrogen receptors and inhibits the growth of human endometrial cancer cells in vitro

Effects of 4‐hydroxytamoxifen, a major metabolite of tamoxifen, on the proliferation of cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in primary culture. Competitive binding studies showed that 4‐hydroxytamoxifen effectively binds to cytoplasmic estrogen receptors (ER) in uterine adenocarcinomas. Of 20 endometrial adenocarcinomas examined, five tumors were successfully grown in primary cell culture. The addition of 4‐hydroxytamoxifen (1 nmol/l to 1 μmol/1) in a medium supplemented with estrogen‐free serum resulted in a dose‐dependent inhibition of the growth of cancer cells in two tumors having ER. However, 4‐hydroxytamoxifen did not affect the growth in the culture system of the remaining three tumors, in which ER were absent in two tumors but were present in one. These results strongly suggest that tamoxifen has a direct growth‐inhibitory effect on human endometrial adenocarcinoma possibly through ER in the tumor.

Divergent effects of prolactin on 4-ene-5α-reductase activity and production of C19-steroids from progesterone in immature rat ovary

Female rats of the Sprague-Dawley strain were used. Two pituitaries from 9-week old rats were grafted in both kidneys of 21-day old rat to induce hyperprolactinemia. All rats with or without pituitary isografts were hypophysectomized on day 26. Starting from day 29, the rats in groups of 8-11 were injected daily with 5 micrograms NIH-LH-S19 or saline for 3 days. Ovarian homogenates from these rats on day 32 were incubated with [14C]4-androstene-3,17-dione or [3H]progesterone and steroid metabolism was estimated. In the hypophysectomized rat ovary, the 5 alpha-reductase activity was stimulated significantly by LH. Although pituitary isograft alone had no stimulative effect on 5 alpha-reductase activity of the hypophysectomized rat ovary, concomitant treatment with LH and pituitary isograft (prolactin) had an additive effect. Formation of the sum of C19-steroids from progesterone in the hypophysectomized rat ovary was stimulated markedly by LH but reduced slightly by prolactin. The LH-induced production of C19-steroids from progesterone was inhibited markedly by prolactin. These results indicate that prolactin treatment inhibits basal and LH-induced production of C19-steroids from progesterone but stimulates LH-induced 4-ene-5 alpha-reductase activity in immature rat ovary.