The pure chromophobes isolated from adenohypophyseal cells of rats were successfully cultured by the roller tube method in a serum-free NCTC-109 medium supplemented with nucleic acids and l-thyroxine. Upon light and electron microscopy, some chromophobes cultured in the control media proliferated by mitosis and others differentiated into immature ambiguous cells. The assay of ACTH, GH, and prolactin showed that these control explants produced a small amount of ACTH and GH, but a large amount of prolactin. When cultured in a CRF (corticotrophin releasing factor) medium, most chromophobes differentiated continuously into acidophils, but not into basophils. Most cells cultured in a CRF medium accumulated granules of various sizes. Since the incorporation of ³H-thymidine into DNA of chromophobes and ambiguous cells reached a maximum at an early stage of their culture, these two cells were assumed to be the maternal cells with the proliferating activity. When the chromophobes were kept in the media containing CRF and colchicine, their cell division was arrested at metaphase. With evident decrease in number of the chromophobes and ambiguous cells, many cells of the explants turned into acidophilic cells providing protein synthesizing ability. Thus the virtual transformation of the chromophobes into acidophils was induced. The explants in a CRF-colchicine medium produced a large amount of ACTH exclusively, and produced neither GH nor prolactin irrespective of the different sizes of granules. During the time course of CRF culture the ³H-thymidine incorporation into nucelar DNA was low when the ACTH content of explants was high, and vice versa. As CRF content of the media was increased, the peak of volumetric percentage of acidophils in the explants appeared earlier. The chromophobes may be dynamic rather than static in a mode of transformation into acidophils following the addition of CRF in to the medium. The cells which were differentiating on the acidophil axis could be, therefore, regarded as ACTH producers in reference to bioassay data.

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Adrenocorticotropic activities of eight octadecapeptide analogues related in structure to the N-terminal sequence of ACTH were compared. The extravascular: intravasuclar potency ratios of three peptides, Ser¹-ACTH (1-18)(I), Ser¹-ACTH (1-18) amide (II) and Gly¹-ACTH (1-18) amide (III), were 1/5-1/8, whereas those of five peptides, β-Ala¹-ACTH (1-18) amide (IV), Ibu¹-ACTH (1-18) amide (V), β-Ala¹, Orn¹⁵-ACTH (1-18) amide (VI), Ibu¹, Orn¹⁵-ACTH (1-18) amide (VII), and β-Ala¹, D-Phe⁷, Orn¹⁵-ACTH (1-18) amide (VIII), were 1-1/2. In vivo half-lives of the steroidogenic and lipolytic activities of the peptides injected intravenously differed slightly, but the ranking of the peptides for both criteria was the same. The in vitro half-lives of the lipolytic activity of peptides, III, IV and V, incubated with fresh plasma were similar and about a half of that of native ACTH, while those of peptides, VI, VII and VIII were much longer than that of native ACTH. When the peptide III was incubated with a medium containing muscle slices, the half-life in lipolytic activity was about 1/6 of that of native ACTH, but those of other peptides were prolonged in the order III<IV<V=VI<VII=native ACTH. These results indicate that (1) an N-terminal β-Ala or Ibu residue resists aminopeptidase activity, which is higher in tissue than in blood, consequently causing a potentiation of the extravascular potency of the peptide.(2) An ornithine residue in the 15th position of the peptide increases the resistance to endopeptidase activity, which is high in circulating blood, consequently prolonging the half-life in plasma in vitro. The relationships between in vitro half-life and two in vitro half-lives in lipolytic activity are considered.

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Time-course studies of ³H-choline incorporation into adrenal phospholipids revealed that the specific radioactivity reached to a maximum level at 9 hr after the isotope injection in untreated control animals, while in ACTH-treated animals, a maximum incorporation was already attained by 2 hr postinjection. The effect of ACTH on the incorporation was relatively prompt, the effect became evident as early as 1.5 hr after the treatment of animals. The rate of the incorporation in ACTH-treated animals was about twice that of the control. The incorporated radioactivity was decreased with the half-life of 2.9 days in phospholipids of control adrenals. On the other hand, the incorporated choline was well retained in adrenals of animals which receive daily treatment with ACTH throughout the experimental period of 7 days. The rate of phospholipid exchange between liver microsomes and adrenal mitochondria was not markedly influenced by treatment of animals with ACTH. Physiological implications of these observations were discussed.

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The effect of implantation of LH (NIH LH Ovine) into the median eminence (ME) area or into the anterior pituitary of ovariectomized rats on pituitary and plasma gonadotropin was investigated. Seven days after implantation, the animals were sacrificed, and FSH and LH activity was determined according to Igarashi-McCann (Igarashi et al., 1964) method and Parlow-McCann-Yokota method (Yokoyama et al., 1965), respectively.
LH implantation in ME significantly decreased plasma FSH levels in two out of the three experiments, and pituitary FSH content in one out of the three experiments. On the other hand, plasma LH levels of LH implanted animals showed a significant decrease in two out of the three experiments. Pituitary LH content of LH implanted animals showed no change in all the three experiments.
LH implantation in anterior pituitary induced no constant change in plasma and pituitary FSH, a significant decrease in plasma LH levels in one and a tendency of decrease in the other two out of the three experiments, while pituitary LH content did not change.
It is concluded that when LH exerts a direct action on the ME, release of FSH and LH is inhibited.
On the other hand, when LH exerts a direct action on the anterior pituitary, release of LH only is inhibited.

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It was found that more than half of the activity of testosterone 5α-reduction was attached to nuclear fraction of rat ventral prostate, and some properties of this reaction were investigated using purified nuclear fraction of the gland.
The optimum pH for reduction of testosterone was about 7.2 and required NADPH as a donor of reducing equivalent. Apparent km of nuclear 5α-reductase was 3.2×10^-5M for testosterone and 3.0×10^-4M for NADPH. Apparent Km for 19-nortestosterone was similar to that for testosterone, but the value for androst-4-ene-3, 17-dione was higher than that for testosterone.
Natural and synthetic estrogenic compounds showed their inhibitory effect on the rate of testosterone 5α-reduction, but one of the gestagen and of anti-androgen did not inhibit the reaction. Estradiol-17β exhibited a most profound inhibition among the compounds examined, and the double reciprocal plots revealed that type of the inhibition is competitive.
The reverse reaction of 5α-reductase using NADP or phenazine dimethyl sulfate as electron acceptor was not detected in purified nuclear fraction from rat ventral prostate.

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The present study was made to determine whether or not an adrenergic relay at the levels of the paraventricular nucleus and of the median eminence participated in regulation of the milk-ejection reflex by utilizing the implantation of dibenamine in lactating rats. In animals bearing dibenamine in the paraventricular nucleus, the litter weight gain on the day following the operation was slightly less than the corresponding value observed in the control animal bearing cholesterol in the same nucleus. The amount of additional milk obtained by the injection of oxytocin was about 2 times higher in the dibenamine implanted animals than in the cholesterol implanted animals, although the difference was not significant. These results suggest that adrenergic nerves terminating at the level of the paraventricular nucleus seem not to play an important role in the regulation of the milk-ejection reflex in rats. The amount of milk obtained by the litters suckling the animals bearing dibenamine in the median eminence was similar to that in the cholesterol implanted controls on the day following the operation. Thus, it is likely that dibenamine in the median eminence did not inhibit the milk-ejection reflex. Systemic administration of dibenamine blocked the milk-ejection reflex. This blockade was overcome by subsequent injection of oxytocin. Therefore, the main site of action of dibenamine to prevent the milk-ejection reflex may occur somewhere along the ascending paths of the suckling stimulus, but not at the level of the paraventricular nucleus, the median eminence and on the myoepithelial cells. The possibility that dibenamine may block the secretion of milk was also discussed.

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The confinement of male rats in complete dankness from birth until 47 days of age resulted in severe retardation of reproductive function. The supply of light-on condition for the last one week caused a remarkable overshoot of testosterone secretion beyond the level of intact animals, associating with a significant weight increase in accessory reproductive organs. Eye-enucleation nullified these effects. This androgen overshoot was considered to be occurred as a result of the removal of inhibitory effect of dark condition rather than a result of positive stimulatory effect of light-on condition.
The possibility to simulate the inhibitory effect of dark condition was examined. The intra-ocular injection of a serotonin precursor (5-hydroxytryptophan) at the initiation of 7 days of light-on treatment, lowered both androgen titer and weight of accessory reproductive organs to the light-off level. This might suggest a possible role of serotonin as an inhibitory dark substance involved in neuroendocrine mechanisms.

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The ¹³¹I-DIT deiodination test revealed a defect of DIT dehalogenase as the cause of euthyroid goiter in a 9 year old boy. Among 21 of his pedigree, 13 had a similar defect of DIT dehalogenase. Four patients with euthyroid goiter, 1 patient with hyperthyroidism, and 8 non-goitrous euthyroid subjects were found among these 13 relatives. No defect of MIT deiodination was found in any of these cases. In this family, the mode of inheritance of the defect of DIT dehalogenase appeared to be autosomal dominant.

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The amygdaloid nuclear complex was stimulated electrochemically in two different types of persistent estrous rats and in castrated male rats bearing an ovarian transplant. In persistent estrous rats induced by continuous illumination, ovulation could be obtained by the electrochemical (EC) stimulation of the medial part of the amygdala (8 out of 12 rats), but the EC stimulation of other areas in the amygdala was ineffective in inducing ovulation. In androgenized rats (treated with 1mg testosterone propionate at 5 days of age), the EC stimulation could not induce ovulation in any of these rats. In addition, ovulation and corpus luteum formation did not occur in ovarian transplants following the EC stimulation of the amygdala in castrated male rats. These results suggest that the amygdaloid influence on the hypothalamic-pituitary gonadotrophic function in lightinduced persistent estrous rats may differ in nature from those in androgenized rats and male rats.

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Effect of hypotension on levels of antidiuretic hormone (ADH) in plasma was investigated in dogs. Mild-to-severe hypotension was induced either by intravenous administration of sodium nitroprusside or by hemorrhage. ADH titers in plasma were measured before and after the induction of hypotension. Marked rises of the concentrations of ADH in plasma were observed following a fall of arterial blood pressure. The physiological meanings of these rises were discussed.

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