On the 20th day of pregnancy in the rat, decapitation, adrenalectomy or thyroidectomy of a fetus was performed in utero. Some decapitated fetuses were given a subcutaneous injection of cortisone acetate or extract of bovine thyroids. On the 22nd day of pregnancy, the fat content of the fetal liver was determined by the weight of crude fat extracted with chloroform-methanol. Remarkable increase of the fat content in the fetal liver followed decapitation. This increase was prevented partially by injected cortisone, but not by injected thyroidal extract. The increase of liver fat, though less than the recorded increase in decapitated fetuses, also followed adrenalectomy. The increase did not follow thyroidectomy. The observations suggest that the effect of decapitation on the liver fat involves in part the adrenal deficiency.

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The secretory cycle of pancreatic islet cells of mice (C₃H), Wistar rats, white rabbits and chickens has been indicated by ultrastructural changes of the roughsurfaced endoplasmic reticulum and Golgi body, both of which represent cellular activity, ranging from the granular to hypogranular appearance of the cytoplasm of pancreatic islet cells. The ergastoplasm seems to be a primary synthetic site of insulin or glucagon molecules and appears to be carried by numerous microvesicles in moderate electron density toward the Golgi zone. These microcarriers then, constitute the lamellar component of the Golgi apparatus. Irregular swelling and shortening of the above lamellae occur and the vacuoles with amorphous raw substance seem to be formed by pinching off at their terminal dilated portions. At the Golgi zone of beta cells of the animals, microfibrils measuring approximately 60Å in width attach on the outer surface of some of the above vacuoles, and occur both inside and through it, whereas in rats, microvesicles may be frequently engulfed into sacs via their incomplete or partially interrupted surrounding membrance. Thereafter, immature granules may develop and gain electron density gradually, followed by a molding of the storage form of characteristic crystallization of mature granules. Moreover, even after leaving the Golgi area the microfilamentous structures also may fall into the sacs of secretory granules in beta cells, although less frequently. The developed granules are released by an emiocytotic process into the perisinusoidal space. A single intravenous injection of glucose or chlorpropamide in the rat was performed and the ultrastructural alterations of beta cell organelles were observed. The primary effect of these two beta cell stimulants were assumed to be different. The granule synthesis, storage and discharge in the stimulated conditions of beta cells were almost the same process as found in the non-stimulated beta cells, except in the maximally effected state of glucose administration.

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The intracellular sites of reactive zinc in cells of the islets of Langerhans, especially beta cells, were observed in rabbits by electron and light microscopy and correlated with granule synthesis, storage and discharge under normal conditions, during prolonged starvation and after dithizone administration. In most normal beta cells the metal was located mainly in mature secretory granules, less on their encasing membranous sacs and often in the intrasaccular empty spaces of developing granules. In a few instances, the metal was located on the endoplasmic reticulum. During 24 days of starvation, the content of histochemically demonstrable zinc in islet beta cells showed a gradual increase as the fasting state proceeded. The ultrastructure of the beta cells in the islets of Langerhans during starvation showed an intense decrease of protein synthesis as it progressed. The granules with distinct electron density began to become fibrillar and/or amorphous and to appear vesicular on the 7th day of starvation. The intracellular zinc began to occupy rather intersaccular cytoplasmic spaces and to appear on the surface of encasing sacs which did not contain the storage form of secretory granules. Near the end of fasting the metal was found in considerable amounts in the intersaccular cytoplasm, as well as on the surrounding membranous sacs. The earliest effect of dithizone was a transient hypergranulation in beta cells followed by the appearance of sacs containing remnants of granules and fibrillar and/or amorphous materials, and finally by disintegration of the surrounding membranous sacs. Various processes of degeneration and necrotization were seen during the first 24 hrs. After 4 days, the beta cells, which were less damaged, appeared hypoactive. The above findings suggest that most of zinc may intrude into maturing granules via their surrounding sacs derived from the Golgi lamellae and form the storage type of secretory granules under interaction of insulin with other combining proteins.

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Endocrine effects on plasma calcium was studied in parathyroidectomized rats. Male Wistar rats parathyroidectomized by hot wire cautery were immediately given various hormonal preparations subcutaneously. In some animals adrenalectomy or thyroidectomy was performed immediately before parathyroidectomy. Blood samples were taken 6hrs. later and the plasma calcium was determined by EDTA titration method. A significant elevation of plasma calcium was observed after thyroidectomy or adrenalectomy, while a decrease in plasma calcium was found after subcutaneous injection of cortisol. No significant change of plasma calcium was seen after subcutaneous injection of ACTH, TSH, GH, lysine vasopressin, LH, corticosterone, noradrenaline, adrenaline, insulin and estrogen. Correlative investigation was further performed to compare the effects of the adrenal and thyroid on calcium metabolism with the emphasis on the hypocalcemic effect of cortisol. It was suggested that the hypocalcemic effect of cortisol was mainly due to the inhibition of intestinal absorption of calcium and not mediated by the secretion of thyrocalcitonin.

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It has been previously shown in our laboratory that glycyrrhizin inhibits the antigranulomatous action of cortisone without affecting the anti-inflammatory action in rats. In the present paper, the work was extended to include the effect of glycyrrhizin on metabolic actions of cortisone in the liver of adrenalectomized rats. Daily injections of cortisone induced a several fold increase of liver glycogen content, tryptophan pyrrolase activity and incorporation of [1-¹⁴C] acetate into cholesterol in rat liver, while concomitant injections of glycyrrhizin blocked these metabolic actions of cortisone. Administration of glycyrrhizin by itself, however, had no effect on the action of cortisone.

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Fractionation of the digest of saliva-parotin-A by Pronase P was carried out by gel filtration on Sephadex G-25 column to give five fractions (P-1--V), using 1M acetic acid as eluant. Of five fractions, only P-I containing peptides of fairly large molecular weights showed potent hypocalcemic and leukocytosis-promoting activities in rabbits. Electrophoretical behavior and amino acid pattern of P-I were quite different from those of saliva-parotin-A.

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Anterior pituitary glands from normal female rats were cultivated by the organ culture method in an attempt to investigate whether extract of the rat hypothalamus may stimulate the glands directly to release FSH, LH or both. Hypothalamic tissues of mature female rats were extracted with 1.0M acetic acid. FSH-RF or LHRF activity of the extract was determined by estimating FSH or LH activity of cultures treated with the extract. FSH activity was measured by the method of Steelman-Pohley, and LH activity was done by the method of Parlow. The extract could stimulate the release of signifocant amounts of LH, whereas brain cortical extract, oxytocin and lysine-vasopressin were inactive. Contamination of LH in the extract was denied. The results provide evidence for the presence of an LH-RF in the rat hypothalamus. On the other hand, whether there is an FSH-RF in the rat hypothalamus needs to be further investigated. Autoradiography of cultivated glands demonstrated that the hypothalamic extract increased the uptake of ³H-leucine by PAS-positive cells, indicating that the action of the extract is probably related to the synthesis of gonadotropin.

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More than 1.0μg of 20α-hydroxypregn-4-en-3-one and progesterone in the ovarian vein blood of rabbits can be quantitatively determined with a new chromatographic system using a partially esterified carboxylic acid type ion exchange resin, Amberlite IRC-50, and a mixture of ethanol and water (3:2 v/v) used as stationary phase and moving phase respectively. Before administration of human chorionic gonadotropin (HCG), no progestin output was observed from the ovary of nonpregnant rabbits. Following administration of HCG, 20α-hydroxypregn-4-en-3-one and progesterone were immediately detected. 20α-hydroxypregn-4-en-3-one was released from the ovary in quantities approximately 12-fold greater than progesterone. Estrogens were not detected. The progestin released from the ovary of pregnant rabbits consisted of 20α-hydroxypregn-4-en-3-one and progesterone. The ratio of progesterone to 20α-hydroxypregn-4-en-3-one was 0.97 at the middle stage of pregnancy. After administration of HCG, 20α-hydroxypregn-4-en-3-one increased significantly, while progesterone remained almost unchanged. The ratio of progesterone to 20α-hydroxypregn-4-en-3-one was 0.19. The presence of 20α-hydroxypregn-4-en-3-one in the ovarian vein seems to imply that this steroid hormone may play a role in the reproductive physiology of rabbits.

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The effects of the gastric and intestinal juice and the liver upon saliva-parotin-A were studied. The biological activities of saliva-parotin-A were stable after the incubation with gastric juice. It was digested by intestinal juice without loss of the hypocalcemic activity, however, its leucocyte activity was destroyed by this treatment.
The hypocalcemic activity was also stable in the liver but the leucocyte activity was decreased slightly after the 1-hour incubation with the liver slice.

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Radioactive iodine labelled saliva-parotin-A (¹³¹I-SPA) was administered to rats by forced feeding or by injection into the ligated intestine in order to study its passage across the intestine. Higher radioactivity localization was found in the kidney as compared to control rats given KI-¹³¹I in the same method. The presence of ¹³¹I-labelled macromolecules in serum was elucidated by the following procedures: phosphotungustic acid precipitation, radio-paper-chromatography and gel filtration with Sephadex G-75. From the results of the present investigation and those reported previously in this proceeding, the possibility of passage of saliva-parotin-A (SPA) across the intestine was raised.

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