A new approach to the preparation of biologically pure human urinary follicle stimulating hormone from human menopausal urine was performed. A crude human menopausal gonadotropin (HMG) with an activity of about 80-90 IU per mg was applied to a column filled with DEAE-cellulose, which had been previously equilibrated with 0.005M ammonium acetate buffer, pH 8.5. By a sodium chloride gradient' method on a variable gradient apparatus, the fraction of 0.08-0.12M sodium chloride concentration had 3 to 4 times higher in specific activity than the starting material. The active fraction was pooled, and was further purified by the gel filtration on Sephadex G-75 and the chromatography on CM-Sephadex C-25. This procedure yielded urinary FSH with an activity of more than 830 IU per mg.
A homogeneous ultracentrifuge pattern was observed with this purified product, however, only faint-stained minor components still remained in the disc gel electrophoresis for further investigation. An isoelectric point of this. product estimated by the electrofocusing method was about pH 4.0. The LH/FSH ratio of our purified urinary FSH was less than 0.1.

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A single injection of 10μg/rat of estradiol benzoate (EB) at 10.00 hr on the day of estrus (designated as Day 1) in 4-day cyclic rats caused a marked increase in ovarian secretion of progesterone and a concomitant decrease in 20α-hydroxypregn-4-en-3-one secretion for over a week. Hypophysectomy on Day 3 completely diminished the secretion of progesterone on Day 5. The treatment with EB induced the surgy releases of prolactin and LH on the evening of Day 2 or 3, but no further release was seen thereafter. In pseudopregnant rats, a marked release of prolactin occurred between midnight and early morning, but any nocturnal release of neither prolactin, LH, nor FSH was seen in the EB treated rats so far examined on Day 4 to 5. Single injections with prolactin, LH and/or FSH on the evening of the first day of diestrus in cycling rats failed to sustain the luteal function. Pretreatment with progesterone, given an hour prior to the EB treatment, completely blocked the release of gonadotropins expected on Day 2 without reducing the elevated secretion of progesterone in the EB treated rats. These results suggest that the gonadotropins released on Day 2 can not be mediators for the luteotropic action of estrogen. Since hypophysectomy after EB treatment reduced ovarian secretion of progesterone to the basal level, it is presumed that estrogen maintains luteal function by acting on the luteal tissue in the presence of hypophyseal factors.

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The in vivo uptake of ³H-progesterone by the hypothalamus and pituitary of the female ovariectomized estrogen-primed rat was studied following an iv. injection of the radiolabeled steroid. ³H-progesterone was preferentially taken up by the pituitary and median eminence of the hypothalamus, but neither by the cerebral cortex nor by the remainder of hypothalamus. In addition, ³H-progesterone taken up by the tissues was mainly distributed in the cytoplasm, but not in the nucleus.
Utilizing Sephadex G-100 column chromatographic analysis, characterization of progesterone-binding protein in the cytosol of the median eminence and pituitary was attempted. The binding protein in both tissues was of macromolecular component with 4.3-7.1S and preferentially bound progesterone, but neither corticosterone nor estrogen. The binding protein was easily saturated by a large amount of unlabeled progesterone, having a limited capacity to bind progesterone.
It has been concluded that the cytosol progesterone-binding protein in the median eminence and pituitary is specific for progesterone with molecular size of 4.3-7.1S and has a limited capacity to bind progesterone.

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Double-antibody solid-phase (Dasp) method was applied to radioimmunoassays for follicle stimulating hormone (FSH) and luteinizing hormone (LH) in serum. The Dasp method uses second-antibody immunosorbent for the separation of free and antibody-bound antigen. Several parameters of the assays including concentrations of antiserum and second-antibody immunosorbent, reaction time and temperature were investigated. Reaction with second-antibody immunosorbent required only 2 hr at room temperature both in the FSH and LH assays. Nonspecific binding of labeled hormone to the immunosorbent was negligible. The FSH and LH assays were specific and sensitive enough to measure FSH and LH in serum. In these studies, the Dasp method proved to be useful for quantifying gonadotropins in large numbers of serum samples.

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Effect of estradiol on hepatic cholesterol synthesis, acetate and mevalonate incorporation into 3β-hydroxysterols in vitro and acetate incorporation in vivo, was examined in relation to the effect of this steroid on plasma and liver cholesterol levels in male rats. A 3-day treatment with estradiol, daily 0.3 mg/rat, caused a decrease in plasma cholesterol level with a concomitant increase in liver cholesterol level and reduced the rate of hepatic cholesterol synthesis from both acetate and mevalonate. Statistical analysis revealed a) significant positive correlations between plasma cholesterol level and the rate of cholesterol synthesis in the liver, and between acetate incorporation and mevalonate incorporation, and b) significant negative correlations between plasma cholesterol level and liver cholesterol level, and between liver cholesterol level and the rate of cholesterol synthesis in the liver. By contrast, a 3-week treatment with estradiol (0.3 mg/rat, twice a week) produced a marked hypercholesterolemia and a significant stimulation of cholesterol synthesis from acetate both in vitro and in vivo, but not from mevalonate. Liver cholesterol levels after chronic treatment were in normal range. These data suggest that the inhibition of hepatic cholesterol synthesis after the acute treatment with estradiol is indirectly caused by the cholesterol accumulated in the liver as the result of cholesterol shifting action of this hormone and the stimulation after the chronic treatment is the effect of estradiol upon cholesterol synthesis to induce hypercholesterolemia.

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Effect of L-5-HTP (L-5-hydroxytryptophane) on the release of human growth hormone (HGH), thyroid stimulating hormone (TSH) and immunoreactive insulin (IRI) was investigated in 7 normal subjects and 25 patients with endocrine disorders. A single oral dose of 200mg of L-5-HTP showed the elevation of HGH in plasma; however, no release of TSH and insulin in plasma of normal subjects. HGH response to L-5-HTP administration was less in quantity as compared with HGH respone to insulin hypoglycemia or arginine infusion. The timing of HGH respone to L-5-HTP was quite variable (between 30 and 120min) in an oral dose of L-5-HTP. No release of HGH by L-5-HTP administration was observed in each of the patients with primodial dwarfism, panhypopituitarism, pituitary dwarfism, and some cases of hypothyroidism. The effect of hyperglycemia or sedative on the release of HGH induced by L-5-HTP was observed. HGH release by L-5-HTP was not observed in these treatments of glucose or diazepam, therefore it might suggest that HGH release by L-5-HTP was inhibited by hyperglycemia or diazepam administration. Suppression of TSH release by L-5-HTP dose was noticed in some patients with hypothyroidism; however no effect in normal subjects. Neither elevation nor reduction of IRI was observed by the L-5-HTP oral dose in normal subjects.
These findings suggested that L-5-HTP might induce the release of HGH from normal pituitary gland, that the release of TSH might be reduced by the dose of L-5-HTP in the patients with hypothyroidism, but not in normal subjects, and that IRI was not affected by the dose of L-5-HTP in normal subjects.

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Antiserum to porcine proinsulin was prepared in a guinea pig. Removal of crossreacting insulin antibodies was achieved by addition of excess porcine insulin. After such treatment, antiserum retained reactivity to porcine proinsulin but lost its ability to bind insulin. Displacement of labeled porcine proinsulin was not observed with 50ng or less of insulin. Both of the synthetic porcine proinsulin C-peptide analogue (62-formyllysine porcine proinsulin sequence 31-63) and the extracted porcine proinsulin C-peptide analogue (sequence 33-63) reacted approximately as well as porcine proinsulin on a molar basis. Cross-reactions between porcine glucagon and bovine proinsulin were not evident.
Almost 100per cent recovery of added porcine proinsulin was noted from talctreated porcine sera. The coefficient of variance was 6.5per cent at a level 10 times the sensitivity for the two assays, and maximum sensitivity of the assay was 20 picogram.
Assayed values were expressed as C-peptide immunoreactivity (CPR), since porcine proinsulin and porcine proinsulin C-peptide exist in peripheral blood, and moreover, porcine proinsulin intermediate forms might be exisist in peripheral blood.
Immunoreactivity of porcine proinsulin in unextracted peripheral blood was assayed in 3 pigs after intravenous glucose load as CPR. Plasma CPR levels at fasting, 30min, and 60min after glucose load were from less than 0.2 to 0.4, 1.65, and less than 0.2 to 0.98ng per ml, respectively.
A specific radioimmunoassay of porcine CPR in unextracted peripheral plasma has been described.

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TRF test with 50-100μg of synthetic TRF was performed in 57 patients with hypothalamic-pituitary disorders. According to the degree and the pattern of TSH response, they were classified as an absent, subnormal, normal or supernormal response and/or delayed pattern of TSH response. 9 of 21 patients with pituitary chromophobe adenoma showed an absent or a subnormal response. Six of them were hypothyroid. 21 of 23 patients with hypothalamic lesions had normal or supernormal TSH response. 5 with a normal response and all of the six with a supernormal response showed clinical or biochemical evidence of hypothyroidism. Since the pituitary of these cases remained intact from tumor invasion, they should be postulated as hypothalamic hypothyroidism. A delayed pattern of TSH response to TRF, where the peak TSH response occurred after 20min, was observed in 17 of 23 in hypothalamic lesions. 6 of 7 patients with diabetes insipidus showed just the same pattern of TSH response as observed in the patients with determined hypothalamic lesions. This delayed pattern of response and normal or supernormal response to a small dose of TRF, such as 50-100μg, could be used to distinguish hypothalamic lesions from pituitary diseases.

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2α, 3α-Epithio-5α-androstan-17β-ol (10275-S) has been known to possess a potent and long-acting anti-estrogenic activity in rodents. In this experiment, 1.25mg 10275-S injected once subcutaneously into the ovariectomized and estradiol-treated mice caused a complete block of vaginal cornification for 24.3 days in spite of the successive treatment with estrogen. Pre-treatment with 10275-S in ovariectomized mice either 4 or 24 hr before tritiated estradiol injection caused significant inhibition of accumulation of radioactivity in the uterus. Pre-incubation of uterine cytosol from immature rat with 10275-S competitively suppressed the formation of estradiol-macromolecule complex maximally around the 8S region, and 50% suppression was obtained at 4.4×10^-7M, about 70-fold over the estradiol. Under the same conditions, 10^-5M progesterone or testosterone gave no effect on estradiol binding at the 8S region. The relation of these in vitro results to the in vivo activities of this steroid was discussed.

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Effects of cortisol and prolactin on the lactation depressive action of estrogen were studied in mice. The daily s. c. injections of 1 erg and 10μg estradiol benzoate (EB) from day 3 of lactation decreased the litter weight gain and caused a regression of mammary glands, pronouncedly with a higher dose and with longer treatments. All young in each litter died between day 6 and 9 of injection in a group receiving 10μg. The twice daily s. c. injections of 0.1mg cortisol acetate significantly reduced such suppressive effects of EB upon lactation. All litters continued to gain weight and could survive throughout the entire period of the treatment (10 days) in cortisol injected groups. But, twice daily s. c. injections of 2.5 I. U. of prolactin could not reduce the depressive effect of EB upon lactation. In Experiment 2, each mouse was isografted with 2 anterior pituitaries (APs) under the kidney capsule on day 2 of lactation and received daily s.c. injections of 1μg and 10μg EB from day 3. The inhibitory effect of EB on litter weight gain appeared to a lesser degree in AP grafted groups than in intact groups in the first experiment. In Experiment 3, each mouse was implanted with a cortisol pellet on one side and a cholesterol pellet on the contralateral side of the inguinal mammary glands on day 2 of lactation and received daily s.c. injections of 10μg EB from the next day. Histological inspection showed that there remained functional alveoli filled with milk only at the cortisol pellet implanted site 10 days after implantation. These results suggest that glucocorticoids may be intimately concerned in the mechanism of lactation inhibitory action by estrogen and counteract, at least in part, directly at the site of mammary tissues, although it is not the only hormone involved in this process.

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In order to elucidate the facilitatory action of progesterone on the CNS mechanism (s) regulating ovulating hormone (OH) release, the steroid (5mg/rat) dissolved in sesame oil was injected subcutaneously to proestrous rats which had been treated with the minimal dose of sodium pentobarbital (PB, 25 or 22.5mg/kg, ip) to inhibit spontaneous OH release. Low (20-30%) incidence of ovulation (No.of rats ovulated against No.of rats treated) was obtained on the following day in the rats given PB alone. When progesterone was injected simultaneously with PB or until 21:00 proestrus, 4hr after PB injection, incidence of ovulation markedly increased to attain about 80 %. In these rats, ovulation was observed from 10:00 to 11:00 on the morning of estrus regardless of the timing of progesterone injection given between 17:00 and 21:00 proestrus. The rats given progesterone at 22:00 or later on proestrus, however, failed to show any increase in the incidence of ovulation. In the rats pretreated with 30mg/kg or more of PB, progesterone was ineffective to increase ovulation incidence. It is likely, therefore, that progesterone exerts its action on the CNS to prolong and possibly heighten the activity to induce OH release. This also suggests that endogenous progesterone, increased by the initial part of released OH on the afternoon of proestrus, acts, in turn, on the CNS ovulating mechanism (s) to accomplish normal OH release.

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The original source of thyroidectomy cells (TX-cells) in rats was investigated electron microscopically. Thyrotrophs did not respond so definitely to thyroidectomy as gonadotrophs did to castration. In thyrotrophs, changes in terms of hyperplasia, hypertrophy and de-or hypergranulation were not manifested. There was no evidence that TX-cells belonged to hyperactive thyrotrophs. The course of transformation from the degranulated acidophils to TX-cells was clearly demonstrated in sequence in this study. Hypertrophy, deformation of the cell, expansion of cisternae, occurrence of intracisternal granules and enlargement of the Golgi-ring, these were manifestations of the developmental process of degranulated acidophils into TX-cells. The other process of transformation of acidophils into TX-cells seemed related to the gradual dilation of ER without discharging their large granules completely. During the chronic phase of thyroidectomy, the anterior pituitaries were filled with TX-cells and their precursors. High vesiculation could be considered as an intrinsic biological reaction which is commonly seen in most of hypophyseal cells during thyroxine deficiency. It was, therefore, suggested that degranulated or slightly granulated acidophils might be predominantly the original sources of TX-cells. Thus, the acidophil theory has been firmly established as to the genesis of TX-cells. We, at the same time, wanted to open up a possibility that some TX-cells might arise from immature basophils, but the valid evidence was not produced in this study. It was verified electron microscopically that the fully developed round TX-cells seemingly like signet-ring-typed castration cells were quite different from genuine castration cells, although both cells had the same staining properties and light microscopical characteristics. However, a number of immature gonadotrophs came into being during the chronic phase of thyroidectomy.

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Serum and pituitary FSH, LH and prolactin contents during the normal rat estrous cycles were assayed every two hours in adult female rats of Holtzman strain (lights on 5 a.m. to 7 p.m.), using NIAMD rat FSH, LH and prolactin radioimmunoassay kits.
The serum LH surge was observed from 6:00 p.m. to 7:00 p.m. on the proestrous day. The serum FSH surge was observed from 7:00 p.m. on the proestrous day to 5:00 a.m. on the estrous day. The serum prolactin concentrations also began to rise at 1:00 p.m. on the proestrous day, and remained at high levels until 10:00 a.m. on the estrous day.
The reduction in pituitary FSH, LH and prolactin content was simultaneously observed from 7:00 p.m. to 11:00 p.m. on the proestrous day.
These results not only comfirmed the data reported by Daane & Parlow (1971), but also demonstrated no diurnal rhythm on the first and second diestrous day. Dissociation between FSH and LH patterns seems to suggest the possibility that FSH and LH secretion may be controlled independently by the two distinct “Releasing factors, FRF and LRF”.

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An increase in protein and both RNA and DNA contents of toad testes was observed following malonate administration. This rise in testicular protein and nucleoprotein contents after malonate treatment was possibly the result or cause of stimulation of both spermatogenic and steroidogenic activities in toad testes.

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The assay of serum progesterone using the competitive protein binding procedure allows rapid and precise measurements of progesterone and makes large population studies on various complications of pregnancy more feasible. Several important factors were found to influence the accuracy and reproducibility of the assay. The levels of serum progesterone in some normal pregnancy, hydatidiform mole and choriocarcinorna are presented.

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Iodoaminoacids or iodide, in a dose containing 50μg iodine stimulated intracellular colloid droplet formation and radioactive iodothyronine release in mouse prepared for McKenzie bioassay. Theophyllin enhanced those effects on both aspects. Those results may explain thyroglobulin-induced enhancement of thyroidal secretion currently drawing attention of many investigators.

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