In this experiment a method of incubating the anterior piuitaries of male rats in vitro was used to study the neurohumoral regulation of hypothalamus on the pituitary. Based on the fluctuation of the gonadotropins upon the addition of crude hypothalamic extract and copper salt in the incubation medium the mechanism of copper-induced ovulation was studied. For the measurement of the gonadotropins the ascorbic acid depletion method of Parlow was used for LH, the rat's ovarian weight measuring method of Steelman-Pohley was employed for FSH, and the methed of measuring the dry weight of pigeon crop sac mucosa of Nicoll for prolactin was employed. The measurement of the gonadotropins in the incubation medium was possible upon selection of an adequate amount of anterior pituitary tissue and the proper incubation time. The gonadotropins from an anterior pituitary in the medium after incubation for 2 hours were in the range of 0.2-0.8μg of LH (NIH-LH-S₁₂), 11-23μg of FSH (NIH-FSH-S₄), and 1.0-44mU of prolactin (NIH-P-B₂), corresponding to conditions of the incubation. The significant increase of the LH and FSH activities and decrease of the prolactin activity after the addition of hypothalamic extract suggest the presence of LH, FSH releasing-factor and prolactin inhibiting-factor. The addition of cerebral cortex of posterior pituitary component caused no such fluctuations. A single addition of the copper-salt produced only an increase of prolactin. The addition of the hypothalamus and copper-salt caused a significant increase of LH and FSH. These results would suggest the action of copper-salt on the hypothalamus to release LH and FSH as the mechanism of the copper-induced ovulation.

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To see whether a periodical central excitation related to the gonadotrophin release or production exists on the other stages than the proestrus in the 4-day cycling rat, the time course of the successful blockade of expected ovulation was investigated with pentobarbital anesthesia applied at different timing of the day throughout the estrous cycle.
Except the day of estrus, the anesthesia delayed the ovulation by one day, when it was given at a more or less limited period on the afternoon of each day of the cycle. Larger doses or more long-lasting anesthetic actions were required on the day of diestrus compared with the days of proestrus and metestrus. It was also noted that the effective period for blocking the ovulation by the anesthesia was much wider on the day of former than the days of latter of which period was almost corresponded with so called “critical period.”
A hypophyseal gonadotrophic activity, assayed by the induction of extra-ovulation in 4-day cyclic mice, showed a sudden change at around 14:00 on the day of metestrus in both rats and mice, though they showed somewhat different pattern.
Existence of some periodical, surging release of gonadotrophin, subjected to the activation of “cyclic release regulating” mechanism in the hypothalumus, is suggested on the day of metestrus in addition to the day of proestrus.

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The role of the pituitary gland on the biosynthesis of corticosteroid-binding globulin (CBG) was investigated in male albino rats. It took 48 hr after adrenalectomy to record a significant increase in corticosteroid-binding capacity (CBC). Likewise, a significant depression of CBC was observed only beyond 48 hr after the start of 40μg/day of dexamethasone injection. From these findings it may be interpreted that glucocorticoids exert an inhibitory effect physiologically upon CBG synthesis and that adrenalectomy would abolish such an depressive action by the elimination of endogenous corticosteroids. Daily administration of 20μg of estradiol resulted in a significant increase in CBC in 5 days. Furthermore, dexamethasone showed inhibiting completely such a stimulatory effect of estradiol on CBC, which was, in contrast, augumented by adrenalectomy. These results suggested that there might exist a competition for the biosynthesis of CBG between the inhibitory action of glucocorticoid and the stimulatory effect of estradiol.
Hypophysectomy did not interfere with the supressive effect of dexamethasone on CBC but abolished the CBC response to adrenalectomy or estradiol treatment, which could not be restored by replenishment with various pituitary hormones, such as ACTH, RGH, RFSH, BTSH or ovine prolactin as well as pituitary autoimplantation. The present results indicate that either unknown pituitary factor (s) or synergic action of multiple pituitary hormones may be needed only when the biosynthesis of CBG is accelerated and elaborated with a mechanism for which the neural and/or vascular connection between the gland and the hypothalamus is indispensable.

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In order to study the effect of calcitonin on the bone collagen metabolism in osteolathyrism, male Wistar rats were placed on 0.1% aminoacetonitrile diet for 9 days. Porcine calcitonin dissolved in gelatin was concurrently administered in a dose of 0.1 MRC U/100g body weight for 8 days and on the 9th day 0.75 MRC U/100g body weight was given 3, 2 and 1hr respectively before sacrifice. Both bone collagen formation and maturation were examined by the determination of specific and total activities of 3Hhydroxyproline in bone collagen, and collagen resorption was examined by the estimation of the amount of hydroxyproline released into the incubation medium from metaphyses. Treatment of lathyritic rats with calcitonin diminished the concentration of serum calcium, phosphate, and rate of collagen resorption, but did not affect collagen formation and collagen maturation. Inhibitory effect of calcitonin on collagen resorption was more pronounced in osteolathyrism than under normal condition. It seemed that parathyroid glands did not play a vital role in the collagen resorption induced by osteolathyrism, since the resorption was not affected by parathyroidectomy. On the other hand, administration of calcitonin (every 0.8 MRC U/rat, 60 and 30min before sacrifice) also counteracted collagen resorption which was induced by low calcium diet and treatment with parathyroid hormone (PTH, 70 USP units).
It is concluded that the effect of calcitonin on collagen metabolism resulted in primary inhibition of collagen resorption in osteolathyrism, and that calcitonin counteracted both PTH-dependent collagen resorption and PTH-independent collagen resorption induced by osteolathyrism.

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Metaphyses and diaphyses removed from rats treated with 0.1% aminoacetonitrile diet for 9 days were incubated separately in Krebs-Ringer bicarbonate buffer. The amount of total hydroxyproline released from diaphyses into medium decreased by parathyroidectomy in lathyritic rats, but not from metaphyses as described previously. Collagen resorption in diaphyses depended more on parathyroid function. The sensitivity for collagenase inhibitors on collagen resorption of diaphyses was different from that of metaphyses. Treatment of lathyritic rats with calcitonin (0.75 MRC U/100g body weight 3, 2 and 1 hr respectively before sacrifice) also resulted in inhibition of the amount of total hydroxyproline released from diaphyses into the medium, as shown in the case of metaphyses. Therefore, it was suggested that calcitonin counteracted the parathyroid hormone-induced collagen resorption in diaphyses.
Calcitonin decreased the amount of total hydroxyproline released from lathyritic metaphyses into the medium, but did not significantly decrease the amount of hexosamine. This result indicated that inhibitory effect of calcitonin on matrix resorption of metaphyses was more selective on collagen than on hexosamine-containing substances. Bone collagenase inhibitors (EDTA and cysteine) also diminished the amount of total hydroxyproline released from lathyritic metaphyses into the medium.
From these results it may be concluded that calcitonin regulates directly or indirectly bone collagenase in osteolathyrism.

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Ion and water movements in the isolated gall bladder of several teleost species were studied in relation to osmoregulation and to hormone treatments. When gall bladders were bathed on both mucosal and serosal sides with isosmotic Ringer solution, a considerable amount of water moved from mucosa to serosa in all the species examined, and sodium and chloride concentrations in the passing fluid were greater than those of the Ringer solution, indicating active transport of these ions. The gall bladder of the freshwater-adapted eel was identical in its ion and water absorption capacity with that of the eel in sea water. No change in this absorption capacity was observed after cortisol treatment of the freshwater eel or prolactin treatment of the seawater eel, although these treatments produced significant effects on ion and water movements in intestinal segments isolated from the same fish. Treatment of gall bladders from starry flounder with oxytocin and arginine vasotocin in vitro showed an inhibitory effect on water movement. Comparative studies of several teleost species reveal no correlation between marine or freshwater existence and the absorption capacity of the gall bladder, nor any relation of the latter to the structure of the gall bladder (tubular or sac-like). The lack of response to environmental salinity changes and to cortisol and prolactin indicates that the teleost gall bladder is not merely an ontogenic extension of the intestinal surface in its osmoregulatory ability.

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Distribution of radioactivity in various tissues has been studied by dry mount autoradiography after the injection of ³H-estradiol in female fetal rats. ³H-estradiol was found to be concentrated in nuclei of cells of the oviduct. Radioactivity is also concentrated in the cell nuclei of the ovary, suggesting some effect of estradiol on these nuclei of cells. In such non-target tissues, as intestine, kidney, lung, liver and muscle, no nuclear concentration of radioactivity was found and average silver grain density per 100μ² is much lower than that of the oviduct. Although, in the adrenal, radioactivity was found to be randomly distributed in cytoplasm as well as in nuclei, the average silver grain density per 100μ² in nuclei was relatively high compared to that of the other non-target organs. It seemed that the estrogen exerts some effect on the adrenal of a fetal rat.

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The effect of accelerating metamorphosis with two different doses of L-thyroxine on the epidermal ATPase activity of Rana catesbeiana tadpoles was examined. Na, KATPase activity progressively increased in epidermis of tadpoles treated with 10ng/ml of thyroxine and at the end of the experimental period the value was around 5 times the control group. In the group of tadpoles treated with 100ng/ml of thyroxine, activity of this enzyme changed more rapidly reaching values 7 times the control on the 10th day of treatment. The effect of thyroxine treatment on epidermal content of Mg-ATPase in tadpoles was considerably less than the changes noted above for Na, K-ATPase; levels after 10 days of treatment were only twice control values. In contrast to its effect in tadpoles, thyroxine treatment effected neither ATPase activity nor oxygen consumption of epidermal tissue from adult Rana pipiens frogs. From these observations it might be concluded that Na, K-ATPase activity of frog skin is specifically dependent on thyroid hormones for its initial activation (during metamorphosis) but not its subsequent control.

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Hypophysial gonadotropin secretion varies in several Salientia inhabiting temperate climates and the sexual cycle in those animals has been correlated accordingly. In experiments conducted on male Rana hexadactyla, a tropical frog with a continuous spermatogenetic cycle, we studied the relation of the hypophysio-gonadal axis to seasons. Pituitary extracts from animals of the summer or winter months were administered to summer or winter frogs and vice-versa. The effects of the extracts were observed on the spermatogenetic cycle of this species. Spermatogenesis was usually accelerated by pituitary extract administered at any time of the year, although the effect was more pronounced in frogs treated with summer pituitary extracts during summer-time than in winter. The probable significance of the results obtained in relation to hypophysial gonadotropin secretion and to the sensitivity of the germinal epithelium of Rana hexadactyla is discussed.

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The increase in weight of the rat ventral prostate after testosterone administration to castrated rats was shown to be accompanied with concomitant elevations of both the rate of testosterone 5α-reduction and that of the incorporation of choline-³H into phospholipids in the glands. Time course study revealed that the increase in rate of 5α-reduction and of choline-³H incorporation became evident by 24 hrs after testosterone administration. The increase in rate of 5α-reduction and of choline-³H incorporation into phospholipids was observed in both microsomal and crude nuclear fractions from castrated rats 24 hrs after testosterone administration. The rate of 5α-reduction and DNA content exhibited the similar recovery after the purification of nuclei, however the radioactivity of choline-³H in the crude prostatic nuclei was lost during the procedure for purification of the nuclei. Therefore, it was concluded that the increase in rate of 5areduction and choline-³H incorporation into phospholipids took place simultaneously after the administration of testosterone, however sites of increased testosterone 5α-reductase did not seem to be located on the newly synthetized membranes.
5α-Androst-l-ene-17β-ol-3-one, known to be a potent androgen, evoked a similar increase in DNA content, the rate of 5α-reduction and DNA polymerase activity after testosterone administration to ventral prostate of castrated rats. This suggested that the increase in testosterone 5α-reduction observed did not seem to be a substrate-induced change.

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Effects of synthetic LH-RH were studied in 5 normal males, 5 normal females and 13 patients with various endocrine or metabolic disorders by measuring plasma LH and FSH levels. In some normal subjects plasma GH and TSH were determined simultaneously with plasma LH and FSH. The administration of 100μg synthetic LH-RH intramuscularly to normal males and females caused a prompt and marked increase in plasma LH levels. The maximum response was observed at the 30 min after the injection and the degree of increments was 6-25 fold in male and 3-6 fold in female. Plasma FSH levels were also increased in 3 male cases and in all female cases, but the degree of responses was smaller as compared with that of plasma LH. A postmenopausal woman with high initial plasma LH and FSH values was responded markedly to synthetic LHRH and the response lasted longer. Synthetic LH-RH had no effects on plasma GH and TSH levels. Patients with pituitary insufficiency failed to show any significant increase in plasma LH and FSH levels in response to synthetic LH-RH. Patients with the other endocrine disorders showed a normal response to synthetic LH-RH as well as 2 cases of anorexia nervosa with amenorrhea. These data indicate that synthetic LH-RH is an useful material to investigate the pituitary gonadotropin reserve in man and that LH-RH has an intrinsic FSH-RH activity, even though it is weaker than LH-RH activity. In addition, the usefulness of this material when employed with the conventional pituitary function tests was discussed.

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The effect of electrical stimulation of the dorsal hippocampus (DHPC) on the concentration of LH, FSH and prolactin in both serum and pituitary gland was studied by chronic preparation in Wistar rats. Electrical stimulation consisting of monophasic square wave pulses of 0.1ms, 100Hz, 60μA was applied for 30min between 10:00 and 14:00 during various stages of the estrous cycle, or between 12:00 and 13:00 on the day of proestrus. The results are as follows: 1) Electrical stimulation of the DHPC did not induce any changes in the serum and pituitary LH levels during estrous cycle. 2) Both serum and pituitary levels of FSH were elevated following the stimulation on the day of estrus, and serum levels of FSH were reduced on the day of diestrus II. 3) Prolactin levels in serum were reduced by the stimulation on the days of proestrus and estrus, whereas pituitary levels were elevated. 4) Stimulation of the DHPC prior to the onset of critical period on the day of proestrus did not affect the ovulatory discharge of LH, FSH and prolactin, however elevated the pituitary levels of prolactin. 5) When stimulation was applied under acute preparation, the DHPC stimulation prevented the ovulation, and also depressed FSH and prolactin as well as LH to be released.
These results suggest that the hippocampus participates in the dual mechanism involved in the release and synthesis of FSH and prolactin.

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The activity of 5α-dihydrotestosterone reduction at 3 positions was mainly observed in the soluble fraction (105, 000×g for 1 hr) from the rat ventral prostate. More than 90% of reduced metabolites were identified as 5α-androstane-3α, 17β-diol when in cubated with various concentrations of the substrate and this may imply that the 3α-reduction of 5α-dihydrotestosterone seems to be a main pathway in this system.
Properties of 3α-reduction of 5α-dihydrotestosterone in the soluble fraction from the rat ventral prostate were examined. Optimal pH of the reaction was around 6.4 and required NADPH or NADH. With the latter nucleotide, however, the rate of reduction was lower than that with NADPH. The reaction showed that it was almost the maximum rate with the concentrations of 5α-dihydrotestosterone over 10^-5M.
The rate of 3α-reduction of 5α-dihydrotestosterone in the ventral prostate was not influenced by castration or testosterone administration. Addition of estradiol-17β to the reaction mixture showed no significant effect on the reaction of 3α-reduction, however testosterone, androst-4-ene-3, 17-dione and cyproterone acetate exhibited an inhibitory effect on the reaction.
The soluble fraction from the rat ventral prostate was incubated in the presence of NADPH and testosterone-¹⁵C, and the formation of androst-4-ene-3D, 17β-diol-¹⁴C was demonstrated.

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Mammary growth was markedly inhibited in mice by a single subcutaneous injection of 100μg hexestrol dicaprylate despite the fact that the pituitary prolactin levels were significantly higher in these mice than in the intact controls. The degeneration of mammary gland was completely counteracted by the simultaneous isologous pituitary grafting, indicating that the additional supply of prolactin from the grafts overcame the inhibitory action of estrogen on mammary growth. These results have demonstrated the existence of antagonistic effects between prolactin and estrogen on mammary growth in mice at some dose levels of estrogen.

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Mammary lactogenic response was obtained in the male rat following a single injection of reserpine (1mg/kg). The lactogenic effect of reserpine was rather long-lasting and the response of the mammaryglands was biphasic. As previously reported (Arai and Suzuki, 1971), the initial peak of the lactogenic response was recognized on day 2, the second one around day 8. Administration of ergocornine (1mg/rat) concurrently with reserpine inhibited the initial phase of lactogenic response when autopsied on day 2. The secondary lactogenic response was also inhibited when ergocornine wasgiven on day 4 or day 6 and autopsied on day 8, but not inhibited when ergocornine wasgiven concurrently with reserpine and autopsied on day 8. These results suggest that ergocornine acts at a different level from that of the lactogenic action of reserpine to prevent pituitary prolactin release, and also suggest that alterations in the hypothalamus caused by reserpine may be primarily responsible for the biphasic release of pituitary prolactin in the male rat.

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Decrease in Type II and concomitant increase in Type III in the hexokinase isoenzymogram of rat carrageenin granuloma were induced in vitro by the addition of insulin to the incubation medium. The changes in the isoenzymogram observed in vitro were quite similar to those induced by insulin in vivo in the same tissue. Addition of pchloromercuribenzene sulfonic acid, a sulfhydryl inhibitor, to the medium also caused decrease in Type II. On the other hand, Type II was markedly increased by the addition of mercaptoethanol, a sulfhydryl compound, to the isoenzyme solution. The findings were discussed with respect to the mechanism of the effect of insulin, especially on the relation between insulin action and state of sulfhydryl groups in the experimental system.

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Glucose tolerances in vivo during perinatal days in the rat were studied. Glucose was injected intraperitoneally, and blood sugar levels were determined 0, 0.5, 1, 2, and 3 hr after the glucose challenge.
The blood sugar level reached the peak at 0.5 hr in all series of animals. In both the premature newborn rats delivered by Caesarean section on the 22nd day of gestation and 2-day-old rats, the levels declined thereafter, but the decline was relatively slower particularly 1 hr after the glucose challenge than that observed in the adult. Glucagon added to glucose did not enhance the glucose tolerance in the young. In pancreatectomized 2-day-old rats and premature newborn rats born to alloxan-treated mothers, the blood sugar level remained high even 3 hr after the challenge.
The results suggest the capacity of the late fetal and early postnatal pancreatic islets for releasing insulin in response to a stimulus of hyperglycemia in vivo.

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