Postnatal development of circadian rhythm of the hypothalamo-pituitary-adrenocortical axis was examined by sacrificing rats twice a day, i. e., 8: 30 a.m. and 6: 30 p.m. Circadian rhythm of plasma corticosterone level became evident during the third week of life. Hypothalamic content of corticotropin-releasing factor (CRF) was not detectable till the end of the second week after birth. A significant increase of CRF activity became manifested in the evening during the third week of postnatal life, whereas the CRF activity in the morning remained undetectable. Thus, it is apparent that a significant circadian rhythm of hypothalamic CRF activity also begins during the third week of postnatal life in the rat under our experimental conditions. The finding may provide further support for the existence of a circadian rhythm in the hypothalamic CRF content with the peak value in the evening and the nadir in the morning.

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The effect of electrical stimulation of the brain on ovulation during estrous cycle in the female Wistar rat was examined by the stimulation of the medial preoptic area using concentric bipolar electrode. Ovulation could be induced 24 hr preceding normal ovulation by the stimulation at the time between 14:00-16:00 on the day of diestrus II. Though the threshold current for induction of ovulation on the day of diestrus II was 5 to 10 times higher than that in the critical period, the numbers of ova in ovulated rats were not significantly different.
The threshold current was gradually decreased from the evening of diestrus II to the morning of proestrus, and showed the lowest value (100-50μA) during the critical period. After the critical period the ovulation was still induced, though the threshold was higher again.
Estrogen (5μg s. c.) injection on the day of diestrus I facilitated ovulation by electrical stimulation at the time between 10:00-12:00 on the day of diestrus II, but not by stimulation at the time between 14:00-16:00.

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Oxytocin in doses of 0.25, 0.5 and 1 I. U. was injected s. c. twice daily for 5 days from day 4 of lactation and the milk ejection response was measured on the last day of injection. The difference of litter weight during 1 hr nursing after 8 hr isolation from the mother was considered as the amount of naturally ejected milk. To obtain the residual milk the litters were allowed to suckle for an additional 30 min after oxytocin in the corresponding doses in each group was injected. Two control groups were similarly injected with 0.1ml of 0.5% chloretone solution twice daily, and to obtain the residual milk single injections of 1 I.U. oxytocin and 0.1ml of saline were given, respectively. As the levels of oxytocin increased, the growth rate of the litter decreased and the % of residual milk increased. In the 2nd experiment, single injections of oxytocin in varying doses from 0.125 to 1 I.U. could recover the % of residual milk substantially to the same extent in groups in which the natural milk ejection had been inhibited by treatments with 1 I.U. of oxytocin twice daily for 5 days. It was shown in the 3rd experiment that in the course of repeated injections of oxytocin, the litter could obtain the milk for only a short period after every oxytocin injection, but they became unable to obtain the milk and lost weight during any other period despite the fact that they vigorously suckled the teats. These results indicate that exogenously administered oxytocin inhibited lactation, primarily through inhibition of the milk ejection in response to normal suckling stimulus.

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Yellow KK mice, carrying the yellow obese gene (A^y), developed marked adiposity and diabetic symptoms in comparison with their control littermates, black KK mice. The blood glucose and circulating insulin levels were increased progressively from 5 weeks of age in yellow KK mice. Age dependent alterlations were also observed in pancreas and kidney. Namely, degranulation and glycogen infiltration of B cells, first observed at 5 weeks of age, were followed by hypertrophy and central cavitation of islets. Renal glomerular changes, which were very similar to diffuse or exudative type of sclerosis in human diabetes, were also recognized in the mice at 16 weeks of age. These changes, though less remarkable, were also noted in their control littermates older than 16 weeks of age. Some metabolic defects were developed, as demonstrated by in vitro experiments. At younger age, lipogenesis by liver and adipose tissue was increased in yellow KK mice, but there was no noticeable difference in glucose oxidation by adipose tissue between both mice. Insulin sensitivity of adipose tissue was decreased with age in both mice, especially in yellow KK mice being reduced more remarkably to its complete loss at 16 weeks of age. These findings indicate that the yellow obese gene not only induces adiposity but also accelerates development of diabetic traits of KK mice. A possible mechanism for the observed diabetogenic action of the gene will be discussed.

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Competitive protein binding (CPB) analysis has yielded an easy and sensitive means for the determination of the steroid hormones in body fluids. We have measured the plasma total corticosteroid (total CS) by the modified Murphy's method, and have developed the simple determination method for plasma 11-deoxycorticosteroid (deoxy CS). Deoxy CS was extracted with a 1: 2 mixture of chloroform and carbon tetrachloride, followed by partition against 3 volumes of water 3 times. The present method requires only one tube for the whole procedure. The recovery rate of the extractioning through partitioning is 66% for 11-deoxycortisol and 4.9% for cortisol. The acuracy of the method is satisfactory, the coefficient of variation is 6-7%. In this method the interference of the steroids other than deoxy CS can not be excluded completely, therefore, the application of this method is limited.
We applied this technique for the evaluation of pituitary ACTH reserve following the overnight administration of metyrapone. After giving each 750mg of metyrapone at 8p. m., 12 midnight and 4a. m., the plasma level of total CS and deoxy CS in normal subjects rose to 10μg/dl or more; on the other hand, in the patients with the impaired ACTH reserve the plasma CS level remained less than 8μg/dl. The deoxy CS value after the overnight metyrapone showed the more distinct difference between normal subjects and the patients with the impaired ACTH reserve. In order to confirm the effect of metyrapone, the additional metyrapone was administered to the patients on one day regular schedule. The plasma CS level did not differ significantly after the one day metyrapone administration. We consider that the overnight metyrapone test with plasma CS measured by CPB method is adequate for the routine clinical use.

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The pituitary gland of the medaka Oryzias latipes consists of three portions: the pars distalis, the pars intermedia and the neurohypophysis. The pars distalis is further divisible into rostral and proximal zones. On the basis of differences in tinctorial and histochemical properties, eight different cell types are distinguished. The rostral pars distalis contains two types of acidophils: one is clearly revealed by acid violet (Type 1, probably cells producing prolactin-like hormone) and the other type is distinguished by lead haematoxylin (Type 2, probably corticotropic cells). The proximal pars distalis contains two types of basophils, probably gonadotropic cells (Type 3) and thyrotropic cells (Type 4), which distinguished from each other by the differences in avidity for dyes, size and location. Further, the proximal pars distalis contains orange G-positive acidophils (Type 5, probably somatotropic cells) and relatively many chromophobes (Type 6). The pars intermedia contains acidophils (Type 7) and basophils (Type 8); the former is lead haematoxylin-and Gomori-positive and the latter is aniline blue-and PAS-positive. The neurohypophysis is roughly divisible into a rostral portion containing mostly Gomori-negative fibers and a caudal portion containing mostly Gomori-positive fibers.

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A case of 18-year-old underdeveloped boy with familial cystathioninuria, goiter, poor hearing and some other physical malformations, i. e. hard palate of a high arch, cubitus valgus, spina bifida occulta and megacolon congenita, is reported. His parents are consanguineous, his maternal grandfather, grandmother and his father are mutually cousins. On a chromosome analysis, No.18 chromosome had a short arm deletion. Cystathioninuria, goiter, poor hearing and retarded growth were favorably treated with administration of desiccated thyroid. A defect of thyroxine formation from iodotyrosine was detected in this case. Therefore, it is suggested that goiter and retarded growth were caused by a relative lack of thyroid hormone in his growth process. Congenital cystathioninuria is a rare disease. Among these cases reported hitherto, the investigations into the relationship between goiter and cystathioninuria have not been made in detail.Experimental results are as follows. L-cystathionine inhibited thyroxine formation from diiodotyrosine, the concentration of 10^-2 M of 1-cystathionine showed an inhibition by about 20% in thyroxine synthesis. Serine dehydrase activity in rat liver was elevated to a strikingly high level following the treatment with triiodothyronine, and decreased markedly following thyroidectomy. Activities of cystathionine synthetase and cystathionases remained unchanged. From the above results, discussion was made on the vicious cycle between the accumulation of cystathionine and the inhibition of thyroxine formation.

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The purpose of this study is to determine the acute effect of morphine on ACTH secretion and to determine whether morphine has a direct action on the adrenal cortex by the adult female Wistar rat. ACTH was measured by the determination of corticosterone levels in adrenal glands, adrenal venous blood and circulatory blood. It should be emphasized that in this paper, mechanism of secretion of adrenal corticosterone by the acute effect (the early stage action) following morphine administration was described. Even in morphinization with five daily morphine (2mg/100g, B. W., i. p.) injections, the account of acute effect was determined immediately after the final injection of morphine administration. Morphine alone can not act as an inhibitor of ACTH release nor as an inhibitor of ACTH release due to histamine stress, and morphinization can not act to inhibit ACTH release or to block the effect of a histamine stress on the secretion of ACTH in the early stage action of the final morphine administration, as evidenced by corticosterone levels both in adrenal glands and in circulating blood of the intact rats. Morphine (2mg/100g, B. W., i. p.) alone caused stimulation of ACTH release as well as the final morphine injection of morphinization, and pretreatment of the rats with morphine (2mg/100g, B. W., i. p.) could not act to block the effect of a histamine (0.5mg/100g, B. W., i. p.) stress on the release of ACTH in the early stage action of morphine administration. A detailed account of the effect of morphine or morphinization with four daily morphine injections on corticosterone levels both in adrenal and in adrenal venous blood of the hypophysectomized-ACTH-treated rat has been reported. Morphine (2mg/100g, B. W., i. p.) alone or with pretreatment of pentobarbital (1mg/100g, B. W., i. p.) does not directly interfere with the response of the adrenal cortex to exogenous ACTH in the early stage action of morphine administration. Morphinization with four daily morphine (2mg/100g, B. W., i. p.) injections in the early stage action following the final morphine injection gave the same results.It seems likely that morphine does not directly act on adrenal corticoidogenesis in vivo. These results suggest in the early stage action of morphine administration that morphine acts to stimulate ACTH release at a higher level than the anterior pituitary and does not directly act on adrenal cortex, and that the acute effect of strong stimuli on the rate of ACTH release is not mediated by a decrease in the concentration of circulating adrenal corticosteroids.

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After the administration of ³H-and ¹⁴C-cholesterol to rats 3 days and 4 hr prior to their death respectively, ester and free cholesterol in adrenal glands were successfully labeled separately with different isotopes. Examination of the ³H/¹⁴C ratio in free and ester cholesterol of adrenal glands and in plasma corticosterone enabled us to study of the role of ester cholesterol in corticosteroidogenesis in ACTH stimulated adrenal glands. By the ACTH administration, the rate of ester cholesterol hydrolysis was increased, while the rate of esterification of free cholesterol was decreased and the hydrolized cholesterol was incorporated into the free cholesterol pool in mitochondria, and then utilized for corticosterone biosynthesis. The administration of cycloheximide did not interfere with the stimulated rate of ester cholesterol hydrolysis in the ACTH-treated adrenals but it prevented the response of plasma corticosterone to ACTH.

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The mechanism of insulin action on hexokinase isozymes was examined by the electrophoresis on cellulose acetate membrane. It is demonstrated that insulin, some (receptor) protein and type II may form a complex sensitive to sulfhydryl compounds, which appears as type III in some tissues, or as the abortive one with disappearance of type II in others. On this mechanism insulin was deduced to act to induce the protein for the complex as well as to form this complex as a constituent.

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