Female rats of the Sprague-Dawley strain were mated 60 days after a single intravenous injection with 5 mg of 7, 12-dimethylbenz (a) anthracene. In order to maintain the lactation for more than 3 weeks, 5 pups (5 to 10 days old) were attached to each mother rat throughout the experiment. Experimental rats were divided into 6 groups : 1. nonlactation (4 rats); 2. 3-week lactation (8 rats); 3. 6-week lactation (10 rats); 4. 8-week lactation (7 rats); 5. 12-week lactation (9 rats); and 6. 17-week lactation (4 rats). Lactating condition was judged from the body weight curves of the 5 pups. The appearance, number and diameter of tumors were recorded every day. The average of the tumor induction time was 140±74 days. The tumors were divided into 3 types : 1. progressive; 2. static; and 3. regressive.
During pregnancy few tumors of the regressive type were noted. However, tumors of the progressive type increased significantly with the weeks of pregnancy. In the first week postpartum, although the total number of tumors was approximately the same as in the last week of pregnancy, the tumors of the progressive type decreased significantly and the tumors of the regressive type increased. In this period most tumors showed a tendency to regress. In the non-lactating group, the tumors of the progressive type began to increase from the fifth to seventh week postpartum without relation to the lactating period (3 to 17 weeks). Vaginal smears were examined every day in all mother rats to determine the recurrence of the estrous cycle. The first estrus appeared 7 days on an average after delivery in the non-lactating group; 27 days on an average in the 3-week lactation group; and 35 to 38 days on an average in the 6-to17-week lactation groups.
Under the condition of high prolactin level, the behavior of the tumors of the progressive type seemed to be related to estrogen. This was also supported by histological findings in the ovaries.

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1) In our previous paper, it was reported that in human endometrium the step of formation of nuclear receptor -estradiol complex was not temperature dependent but in rat uteri temperature dependent.
In order to clarify whether this temperature dependency in rat uteri means an energy requirement for this step or not, experiments with 2, 4 dinitrophenol as an energy uncoupler were done. Even when rat uteri or endometrium of women were incubated with 10 μc of ³H-estradiol and 10⁴M of 2, 4-dinitrophenol, 8S estrogen receptor-E2 complex in cytosol and 5S estrogen receptor-E2 complex in nuclear extract were recognized in both rat and woman. Namely, no evidence was recognized for this step to require activation energy in rat uterus as well as in human endometrium.
2) The cytosol fraction was prepared after human endometrium had been incubated with ³HE2 at 2°C. Cytosol was analysed on sucross density gradient after heat treatment (at 10°C, 31°C or 37°C). At 31°C and 37°C treatment, the 8S receptor peak was difficultly recognized. Namely, the 8S receptor-E2 complex was heat labile in cell free condition. This appears to be one of the reasons why 8S cytosol receptor was not visible in human endometrium incubated with ³HE2 at 37°C.
3) When the cytosol fraction from the human endometrium was analysed on 3-20% linear sucrose gradient containing 0.4M KCl, the 8S peak diminished and about 4S peak appeared. This was considered to mean that 8S receptor in human endometrium was also split to a 4S binding unit at high salt condition and the 8S receptor in humans had also a subunit structure.
4) 8S receptor in cytosol and 5S receptor in nuclear fraction were recognized in one case of human endometrial carcinoma, but in another case both receptors were not recognized.
5) Estrogen binding protein in human serum was sedimented at 6S fraction by sucrose gradient.

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In an attempt to detect the existence of histaminergic mechanisms in the regulation of prolactin secretion in rats, the effects of histidine and histamine-receptor antagonists (chlorpheniramine and metiamide) on plasma prolactin levels in urethan-anesthetized rats were investigated in relation to other aminergic mechanisms. Chlorpheniramine was used as an H1-receptor antagonist and metiamide as an H₂-receptor antagonist.
Wistar male rats were used under urethan anesthesia and blood was obtained by cardiac puncture. Rat plasma prolactin was measured by radioimmunoassay.
As already reported, L-DOPA and diethyldithiocarbamate markedly decreased plasma prolactin levels in rats. Dihydroxyphenylserine increased prolactin levels. Brocresine phosphate, a histidine decarboxylase inhibitor, markedly stimulated prolactin secretion in urethan-anesthetized rats. This stimulation was not blocked by pretreatment with histidine or L-DOPA. Histidine alone did not affect plasma levels of prolactin or methyldopamine-induced increase in plasma prolactin levels. On the other hand, histidine significantly stimulated prolactin secretion in chlorpheniramine-treated rats. In contrast, histidine depressed plasma prolactin levels in metiamide-treated rats.
These findings indicate that a dopaminergic component is inhibitory and a noradrenergic one is stimulatory in prolactin secretion in rats and further that there exist the histaminergic mechanisms, relatively independent from other aminergic mechanisms, which might also play an important role in the regulation of prolactin secretion in rats. Furthermore, it is suggested that H₁-receptor is stimulatory and H₂-receptor is inhibitory in prolactin secretion in rats.

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It is known that Prostaglandins (PGs) have many interesting physiological effects on various tissues, but the reducing effect on circulating eosinophils was still obscure, except for our report in 1975. Our previous report revealed that the effect had not act through β-adrenergic receptors, because a β-adrenergic blocker did not inhibit this effect.
The present report was carried out to study whether the hypothalamo-hypophysealadrenocortical axis would be concerned or not on this effect.
PGs (10 μg/kg) were administered intraperitoneally under thiopental sodium anesthesia and blood was drawn from the tail vein of the splenectomized rats.
Both PGE₁, E₂, F_2α a did not decrease the number of peripheral eosinophils on the group of the adrenalectomized rats.
Both PGE₁, F_2α a did not decrease the number of peripheral eosinophils, but PGE₂ had a significant reducing effect on the number of them 2 and 3 hours after administration in the group of the rats with hypothalamic lesions.
Both PGE₁, E₂, F_2α had a significant reducing effect at the group of the rats with man-made hypothalamic lesions. This effect was almost the same as for the control group just splenectomized.
It could be concluded that the eosinopenic effect of PGE₁and PGF_2α was conducted through the hypothalamus and adrenal glands, but that of PGE₂ might be effected through the hypophysis or directly through the adrenal glands.

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To study the effect of vatious amines on TRH contents in rat brain, various amines or inhibitor of synthesis of amines were injected into rat through i.v. or i.p.. Rats were decapitated and brain was frozen in dry ice and aceton. TRH contents in hypothalamus (H), cerebrum (C) and cerebellum and brain stem (C and S) were measured by TRH radioimmunoassay.
TRH contents in normal rats were 3.9±0.5ng in H, 2.6±0.5ng in C and 1.6±0.3ng in C and S. TRH contents in all parts of brain were increased in L-DOPA treated group and did not change in T3 or T4 treated group. TRH contents in all parts of brain were decreased in α-methyl-DOPA, α-methyl-para-tyrosine, fusaric acid and 5-HTP treated groups. In D, L-p-chlorophenylalanine treated group TRH contents in brain were increased only in hypothalamus. In L-DOPA or 5-HTP treated group with T4 or T3 preadministration, TRH contents in all parts of brain were same levels of L-DOPA or 5-HTP treated group.
The above data suggested the TRH contents in rat brain were increased with increase of dopamine level in rat brain and decreased with increase of serotonine level or decrease of noradrenaline level in rat brain and inhibitory effect of T4 or T3 on TRH release might be mediated through dopaminergic and serotonergic mechanism.

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Radioimmunoassay of rat TSH was established with purified rat TSH for iodination, standard rat TSH and anti-rat TSH supplied by NIAMDD. iodination of rat TSH was performed with the method of Greenwood et al. This assay was successful in application to determine the plasma and pituitary TSH level in rats. In this assay the minimum detect-able level was 10 ng/tube of NIAMD-Rat TSH-RP-1. Neither rat GH, prolactin, LH, nor synthetic ACTH interferred with the binding of rat TSH to anti-rat TSH. The recovery rate of TSH added to rat plasma was 80-114%.
Intraassay and interassay coefficients of variation were 3.45-7.83% and 3.14-11.33% respectively.
Plasma TSH level was 0.47±0.04 μg/ml in intact male adult rats and 0.35±0.03 μg/ml in pentobarbital-anesthetized ones.
Plasma and pituitary TSH levels after thyroidectomy were investigated every second day during 2 weeks. The former gradually elevated until 2 weeks after operation, while the latter, 290±30 big/mg gland before thyroidectomy, were reduced to 70±21 μg/mg gland on the 4th postoperative day, and thereafter restored to a certain degree. The changes of plasma TSH level immunoassayed in thyroidectomized rats paralleled those obtained by the bioassay within their detectable ranges.
Cold exposure for 60 minutes at 0±1°C resulted in the increasing plasma TSH from 0.35±0.05 μg/ml to 0.97±0.06 μg/ml. After the cessation of cold exposure, plasma TSH levels were restored to the resting levels within 60 minutes.
Intravenous administration of synthetic TRH over the range from 0.5 ng to 500 ng resulted in a rapid plasma TSH increment with peak response between 6 and 12 minutes. Minimum effective dose of TRH on TSH release was 0.5 ng.
The effect of aging was examined, revealing that plasma TSH levels were 0.35±0.03 //g/m1 and 0.74±0.07 μg/m1 at 7-9 weeks and 120-150 weeks after birth respectively, while pituitary TSH were 292±17 μg/mg gland and 66±13 μg/mg gland in the corresponding week with age respectively. TRH-induced TSH response in the aged rats was attenuated and delayed.

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A radioimmunoassay for plasma ACTH has been developed utilizing highly purified human ACTH (Li) labelled with ¹²⁵I by the lactoperoxidase method as a tracer and an ACTH antibody produced by immunization of rabbits with ACTH-Z (Organon). The assay is highly specific, reproducible and sensitive to 20 pg of ACTH per ml. Utilizing this technique, endogenous ACTH secretion, adrenal responsiveness to endogenous ACTH and hypothalamic pituitary adrenal feedback mechanisms have been assessed in normal subjects and in patients with Cushing's syndrome due to adrenocortical hyperplasia and nodular cortical hyperplasia.
The potential effect of a negative feedback mechanism on the circadian rhythmicity after SU-4885 administration was assessed by initiating SU-4885 either at 9 p.m. or 8 a.m. In normal subjects, the circadian rhythm of ACTH was persistent and independent of a decrease in cortisol. However, in a single case of Cushing's syndrome due to adrenocortical hyperplasia, the circadian rhythm was different from that of normal subjects, possibly influenced by a negative feedback mechanism when SU-4885 was initiated at 8 a.m.
In Cushing's syndrome due to adrenocortical and nodular cortical hyperplasia, a significant correlation was observed between the mean plasma ACTH and urinary 17-OHCS values before and after SU-4885 administration (r=0.743, p<0.01). A significant correlation was also obtained in normal subjects between plasma ACTH and urinary 17-OHCS values (r=0.889, p<0.01). However, there was quantitatively more 17-OHCS excreted in the urine for a given plasma ACTH level in patients with Cushing's syndrome than in normal subjects. To assess the relative biological activity of endogenous and exogenously administered ACTH, the ratio of daily 17-OHCS during SU-4885 administration and Cortrosyn-Z administration was expressed as the Cortrosyn Equivalent Quotient (C.E.Q.).
The correlation between plasma ACTH and C.E.Q. was similar and significant for normal subjects and patients with Cushing's syndrome (r=0.670, p<0.01). These data suggest that there is hyper-responsiveness of the adrenal glands to endogenous ACTH in Cushing's syndrome due to adrenocortical hyperplasia and nodular cortical hyperplasia and that the adrenal hyperactivity is not engendered by a qualitative change in the ACTH release from the pituitary gland.
To assess pituitary suppressibility, dexamethasone was administered 40 days or more later following total adrenalectomy in 9 patients with Cushing's syndrome, 6 with adrenocortical hyperplasia and 3 with nodular cortical hyperplasia. One day after discontinuation of substitution therapy, 2 mg of dexamethasone was administered orally followed on successive days by 4 and 8 mg doses. In each instance, dexamethasone was given at midnight and the plasma ACTH concentration was determined at 9 : 00 a.m. on the day before and after administration of the dexamethasone. A patient with Addison's disease was studied as a control. The suppressibility of ACTH secretion by dexamethasone was poorer in Cushing's syndrome due to adrenocortical hyperplasia than in the control and the degree of suppressibility was unrelated to the duration of therapy which ranged from 74 to 3132 days. This suggests that the impairment of negative pituitary adrenal feedback is persistent even after surgery. In nodular cortical hyperplasia, feedback mechanisms could not be assessed post-operatively since the plasma ACTH levels prior to dexamethasone administration were quite low in comparison with hyperplasia group, presumably as a result of depletion of pituitary ACTH due to continued pituitary suppression following adrenalectomy.
In conclusion, these observations suggest that both adrenal hyper-responsiveness to ACTH and impaired negative hypothalamic pituitary adrenal feedback mechanisms are present in Cushing's syndrome due to adrenocortical hyperplasia.

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The effects of the oral contraceptive on the pituitary content and plasma level of LH and the hypothalamic LH-RH level were investigated in 16 adult female rabbits. The oral contraceptive preparation, Sophia-C (Norethindrone 2mg + Mestranol 0.1mg), as administered orally by a stomach tube each day for 7 days in 8 adult female rabbits. At the end of the treatment, the rabbits were bled from the abdominal aorta into heparinized syringes and the plasma was separated. The stalk median eminences were excised. All the materials were stored in-80°C until assayed.
Plasma level and the pituitary content of LH and the hypothalamic LH-RH were measured by radioimmunoassay. All the dose response curved were drawn using logit-log transformation.
Radioimmunoassay procedures for LH was described in detail elsewhere. Purified rabbit LH for iodination (125-I) and standard were prepared by T. Makino and R.O. Greep, at Research Laboratories for Human Reproduction, Harvard Medical School, Boston, U.S.A. The starting B/T ratio was 25% at the final dilution of the antibody of 1/20,000. Minimal detectable quantity was about 40 pg/tube. The 50% intercepts were approximately 460 pg/ tube.
Radioimmunoassay procedures for LH-RH were performed according to the method described by Arimura et al. Antiserum against synthetic LH-RH was kindly supplied to us by Drs. A. Arimura and A.V. Schally, New Orleans, U'S.A. The synthetic LH-RH was kindly supplied to us by Dr. N. Yanaihara. The starting B/T ratio was 29% at the final dilution of the antibody of 1/17,500. Minimal detectable quantity was about 40pg/tube and the 50% intercepts were 150pg/tube.
It has been assumed that oral contraceptive drugs exert their action by blocking the hypothalamic LH-RH, resulting in a depression of the plasma level of LH, because plasma level of LH returned to the normal level when LH-RH was administered intravenously even while oral contraceptive steroids were given continuously. However, these findings concerning the site of action of the drugs furnished only indirect evidences. The possibility of a direct inhibitary effect of these steroids on the anterior pituitary cannot be ruled out.
Administration of 2mg of Norethindrone and 0.1mg of Mestranol significantly depressed both the pituitary content and the plasma level of LH. On the other hand, they significantly increased the hypothalamic LH-RH level. Those findings strongly suggested the direct action of this drug on the anterior pituitary in the female adult rabbits.
Detailed mechanisms on how the hypothalamic LH-RH was increased were not known in this experiment, further investigations are now in progress.

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