A number of conditions related to sex-reversal in boys and men and precocious puberty in girls are caused by estrogen-secreting adrenal tumors. In these tumors, cytochrome P450 aromatase (aromatase) that is encoded in the CYP19 gene is expressed at high levels. To investigate the molecular mechanism of aromatase expression in these adrenal tumors, we characterized the activity, gene transcript and genomic promoter region of aromatase in the human adrenocortical carcinoma cell line H295R. Aromatase activity and the transcript of the CYP19 gene were highly up-regulated by forskolin, but not by dexamethasone. The results from exon I-specific reverse transcriptase (RT)-PCR and the transfection of reporter constructs suggested that promoter I.3 and promoter II were activated in H295R. Deletion and mutation analysis suggested that cAMP response element-like sequence (CLS) and steroidogenic factor-1 (SF-1) motif, were critical for the activation of promoter II. The results of this work should provide the basis for the molecular analysis of aromatase expression in adrenocortical cells.
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J. Watanabe, S. Kanamura, K. Kanai, M. Asada-Kubota, and M. Oka
ABSTRACT
The role of microtubules in the regulation of glucagon receptors on cultured rat hepatocytes was studied. Antimicrotubular reagents, colchicine and vinblastine, did not affect the binding of 125I-labelled glucagon to hepatocytes at 4°C. At 20 and 37 °C, however, the reagents reduced the binding after 60 or 90 min of incubation. Scatchard analysis indicated that the reduction in the binding was due to loss of glucagon-receptor populations. If hepatocytes were preincubated with both unlabelled glucagon and the reagents at 37 °C, the binding of the ligand to the cells decreased markedly after a certain delay. The reagents did not inhibit the internalization of the ligand in the cells until 30 min of incubation at 37 °C. The results suggest that the microtubule system plays a role in the transport of glucagon receptors to the plasma membrane, which is followed by their internalization.
J. Endocr. (1985) 106, 125–131
T. Endo, H. Watanabe, H. Yamamoto, S. Tanaka, and M. Hashimoto
ABSTRACT
While prostaglandin F2α (PGF2α) has been thought to be a natural luteolysin in non-primates, a luteolytic effect in the human corpus luteum is less evident. We therefore investigated the action of PGF2α on monolayer cultures of human luteal cells obtained from mid-luteal phase corpora lutea.
PGF2α increased basal and human chorionic gonadotrophin (hCG)-stimulated progesterone production by human cultured luteal cells. A potent tumour-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), also stimulated progesterone production by cultured human luteal cells.
Although human luteal cells were incubated for 24 h with PMA, hCG was still able to stimulate the production of progesterone by PMA-pretreated cells. However, PMA pretreatment blocked the ability of PGF2α to stimulate progesterone production. It is possible that the luteotrophic effect of PGF2α may be mediated, in part, by the activation of protein kinase C.
Addition of PGF2α to suspensions of human luteal cells preincubated with myo-[2-3H]inositol promoted an increase in labelled inositol phosphates. PGF2α also rapidly increased intracellular free Ca2+ in human luteal cells loaded with the fluorescent Ca2+ probe, fura-2.
We conclude that PGF2α and PMA stimulate progesterone production and that PGF2α increases the intracellular free calcium and inositol phosphates of human cultured luteal cells in the mid-luteal phase.
Journal of Endocrinology (1992) 133, 451–458
S Asai, R Ohta, M Shirota, G Watanabe, and K Taya
The high- and low-avoidance animal (HAA and LAA respectively) strains of Hatano rats were originally selected and bred from Sprague-Dawley rats for their performance in the shuttle-box task. The present study focused on the activity of the hypothalamo-pituitary-adrenocortical (HPA) axis of HAA and LAA rats in response to restraint stress. The restraint stress induced an elevation in plasma concentrations of ACTH, prolactin, corticosterone and progesterone. Peak levels of plasma ACTH during stress conditions were significantly higher in HAA rats than in LAA rats, while peak levels of prolactin were significantly lower in HAA rats than in LAA rats. Under stress conditions, ACTH and prolactin synthesis in the anterior pituitary glands was significantly higher in HAA rats compared with LAA rats. The peak plasma concentrations of corticosterone, during restraint stress, were significantly higher in LAA rats compared with HAA rats. These results indicate that the response of the hypothalamo-pituitary axis to acute restraint stress is greater in HAA rats than in LAA rats, whereas the ACTH-induced adrenal response of corticosterone release is higher in LAA rats than in HAA rats. On the other hand, prolactin secretory activity is higher in LAA rats compared with HAA rats. These differences in endocrine responses to stress may be involved in the regulation of the avoidance responses in the shuttle-box task.
K Ohshima, H Kishi, M Itoh, G Watanabe, K Arai, K Uehara, NP Groome, and K Taya
Plasma concentrations of inhibin pro-alphaC, inhibin A and inhibin B were determined by enzyme-linked immunosorbent assay at 6 h intervals throughout the 4-day oestrous cycle of the golden hamster. Plasma concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta were also measured by radioimmunoassay during the oestrous cycle. Plasma concentrations of inhibin A increased from the early morning of day 1 (day 1=day of ovulation) and reached plateau levels at 0500 h on day 2. An abrupt increase in plasma concentrations of inhibin A was found at 1700 h on day 4, when the preovulatory FSH surge was observed. An increase in plasma concentrations of inhibin B occurred on day 1 and reached plateau levels at 1700 h on day 1. The levels remained elevated until 0500 h on day 4 and declined gradually by 2300 h on day 4. Plasma concentrations of inhibin pro-alphaC gradually increased with some fluctuation from day 1 to 1700 h on day 4 and then declined. Significant negative relationships were noted between plasma FSH and both dimeric forms of inhibin from day 1 to day 3. Significant positive relationships were found between plasma oestradiol-17beta and inhibin A or inhibin pro-alphaC throughout the oestrous cycle. In contrast, no significant relationship was found between plasma oestradiol-17beta and inhibin B. These findings suggest that both dimeric forms of inhibin play a role in the regulation of FSH secretion during follicular development. These findings also suggest that inhibin pro-alphaC could be secreted primarily by large follicles, and early atretic follicles could also be responsible for inhibin pro-alphaC secretion. On the other hand, the secretory pattern of dimeric inhibins might shift from inhibin B to inhibin A with follicular development.
H Kishi, T Okada, M Otsuka, G Watanabe, K Taya, and S Sasamoto
Abstract
The present study was conducted to study the effect of immunoneutralization against endogenous inhibin on FSH, LH, oestradiol-17β and progesterone secretion and to investigate the effect of removal of endogenous inhibin on subsequent follicular development in the hamster. After treatment with anti-inhibin serum (inhibin-AS) at 1100 h on day 2 of the oestrous cycle (day 1=day of ovulation), a marked increase in plasma levels of FSH and a slight increase in plasma levels of LH were noted and pituitary contents of FSH, but not LH, were also increased. In the group treated with inhibin-AS, superovulation occurred on day 1 of the following cycle. Plasma levels of oestradiol-17β markedly increased with the increase in the number of ovulations induced by human chorionic gonadotrophin (hCG) as compared with those in control animals. In the second cycle, plasma concentrations and pituitary contents of FSH in the animals given 200 μl inhibin-AS still showed high values as compared with those in the animals treated with control serum, although superovulation did not occur on day 1 of the third cycle. Plasma concentrations and pituitary contents of LH in the hamster given 200 μl inhibin-AS tended to decrease as compared with those in control animals during the second cycle. Plasma concentrations of oestradiol-17β in the animals treated with 200 μl inhibin-AS changed in a similar way to controls. A marked increase in plasma concentrations of progesterone was noted on days 1 and 2 of the second cycle in the group receiving inhibin-AS. The twice daily injection of 1 IU hCG during the second cycle to the animals given 200 μl inhibin-AS induced superovulation on day 1 of the third cycle.
These results indicate that circulating inhibin may be an important indicator of the number of developing follicles and may maintain the species-specific number of developing follicles through suppression of FSH secretion in the cyclic hamster. They also suggest that high levels of inhibin slightly suppress plasma levels of LH, indicating that plasma LH may also regulate follicular development in the cyclic hamster.
Journal of Endocrinology (1996) 151, 65–75
H. Kaneko, M. Yoshida, Y. Hara, K. Taya, K. Araki, G. Watanabe, S. Sasamoto, and Y. Hasegawa
ABSTRACT
To investigate the physiological importance of inhibin in the regulation of FSH secretion in prepubertal bulls, animals (6-month-old) were passively immunized against inhibin. Five animals were given an i.v. bolus injection of 50 ml inhibin antiserum raised against bovine 32 kDa inhibin in a castrated male goat, and four bulls were given the same amount of castrated male goat serum (control serum) as controls. Treatment with the inhibin antiserum resulted in a marked increase (P < 0·01) in plasma concentrations of FSH within 12 h compared with control animals, and FSH levels in immunized animals remained high until 168 h after the injection. Concentrations of plasma LH and testosterone in the immunized animals were not different from those in the control animals. The present findings provide strong evidence that inhibin plays an important role in the inhibitory regulation of FSH secretion in prepubertal bulls.
Journal of Endocrinology (1993) 137, 15–19
K. Shinzawa, S. Ishibashi, M. Murakoshi, K. Watanabe, S. Kominami, A. Kawahara, and S. Takemori
ABSTRACT
In an attempt to elucidate the functional role of the three zones of the adrenal cortex, the localization of microsomal cytochrome P-450s (P-45017α,lyase and P-450C21) and their relation to steroidogenesis in the guinea-pig adrenal cortex were studied. The intracellular localization and zonal distribution of P-45017α,lyase and P-450C21 were examined by the direct peroxidase-labelled antibody technique. The cytochromes were localized immunocytochemically on the smooth-surfaced endoplasmic reticulum. P-45017α,lyase was found to be distributed in the zona fasciculata, especially in the externa, and less in the zona reticularis. The zona glomerulosa was negative for immunohistochemical staining for P-45017α,lyase. In contrast, P-450C21 was distributed in all three zones in the adrenal cortex.
The cytochrome P-450 contents and their steroidogenic activities were determined with microsomes prepared from the outer zone (zonae glomerulosa and fasciculata) and from the inner zone (zona reticularis) of guinea-pig adrenals. Using an enzyme-linked immunosorbent assay, the ratio of P-450C21 content in the inner zone to that in the outer zone microsomes was estimated to be 1 ·24. 21-Hydroxylase activity was higher in the inner zone microsomes, which was in good agreement with the relative P-450C21 contents. The ratio of P-45017α,lyase content in the inner zone to that in the outer zone microsomes was estimated to be 0·75. 17α-Hydroxylase and C17–20-lyase activities were five- to sixfold greater in the outer than in the inner zone microsomes, which could not be explained by the P-45017α,lyase contents in the two zones.
The effects of ACTH on the zonal distribution of microsomal cytochrome P-450s and their steroidogenic activities were also investigated.
J. Endocr. (1988) 119, 191–200
T. Hamada, G. Watanabe, T. Kokuho, K. Taya, S. Sasamoto, Y. Hasegawa, K. Miyamoto, and M. Igarashi
ABSTRACT
A sensitive radioimmunoassay (RIA) for the determination of inhibin in peripheral plasma and tissue homogenates of different species has been developed using antisera to partially purified bovine follicular fluid (bFF) inhibin and 125I-labelled bFF 32 kDa inhibin. Antisera were produced by immunization of rabbits with partially purified bFF inhibin prepared by immunoaffinity chromatography. Increasing doses of a high titre antiserum could neutralize the suppressing effect of bFF, porcine follicular fluid and rat ovarian homogenate on FSH secretion from rat anterior pituitary cells in culture. Sensitivity of the assay was 3·1 ng International Research Standard of porcine inhibin per tube. Parallel inhibition curves were obtained for inhibin preparations from female and male animals of ten species, i.e. cattle, goats, sheep, cats, dogs, monkeys, pigs, horses, rats and man. Inhibin subunits and related proteins cross-reacted minimally with the antiserum used in the study. Plasma concentrations of inhibin in adult male and female rats were measured by the RIA before and at various times after gonadectomy. Inhibin levels in peripheral plasma before gonadectomy were significantly higher in adult female than in adult male rats. Inhibin levels decreased abruptly after gonadectomy in both sexes and they correlated negatively with plasma concentrations of FSH. This inhibin RIA will facilitate studies of the physiology of inhibin in various species of animals.
Journal of Endocrinology (1989) 122, 697–704
S Asai, R Ohta, M Shirota, A Tohei, G Watanabe, and K Taya
Hatano high-avoidance (HAA) and low-avoidance (LAA) animals were originally selected from Sprague-Dawley rats for good and poor active avoidance learning in a shuttle box. We studied the endocrinological profile in lactating rats to determine the effect of suckling during mid-lactation in HAA and LAA rats. The pups were separated from their mother rats 6 h before the onset of suckling and blood samples were drawn from unanaesthetized mother rats via a jugular cannula at 0, 5 and 15 min after the suckling stimulus and then 15, 45 and 105 min after pups were removed. Plasma concentrations of oxytocin in HAA rats were significantly higher than in LAA rats during the suckling period. Plasma concentrations of prolactin and ACTH in HAA rats were significantly higher than in LAA rats during the suckling period, and at 15 min and 45 min after the pups were removed. However, there were no strain differences in circulating corticosterone between the two lines, indicating that the response of the hypothalamo-pituitary axis to the suckling stimulus was greater in HAA rats than in LAA rats, whereas the ACTH-induced adrenal response of corticosterone release was higher in LAA rats than in HAA rats. Since dopamine from the median eminence inhibits prolactin secretion from the lactotrophs of the anterior pituitary, and tuberoinfundibular dopaminergic neurones are partially regulated by the level of circulating prolactin, we evaluated the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. TH, measured by the accumulation of 3,4-dihydroxyphenylalanine, was significantly higher in HAA rats than in LAA rats before the suckling stimulus. After the suckling stimulus, TH activity in HAA rats was significantly lower than before suckling, whereas TH activity in LAA rats was not changed. These findings clearly demonstrated that apparent differences between the two Hatano lines exist in endocrinological profiles during suckling. These strain differences probably originate from neurotransmitter changes, such as dopamine.
