Partial purification of growth hormone (GH)-releasing factor (GRF) by acid extraction followed by gel filtration on Sephadex G-25 has been attained from bovine hypothalami. When rat pituitaries were incubated in 2ml Krebs-Ringer-bicarbonateglucose (KRBG) medium, a stimulatory effect of the GRF fraction on immunoreactive GH (IR-GH) release was observed, while that of the factor neither on GH synthesis nor release of the synthesized GH was demonstrated. Stimulation of the GH release was exerted maximally within 30min of incubation. Cycloheximide and actinomycin D, at a concentration which inhibited protein and RNA synthesis to less than 5 and 20% of the control, respectively, were without effect on the stimulatory action of the factor on GH release. On the other hand, stimulation of GH synthesis was observed under incubation in 0.3ml medium with the factor and enhancing effect of the factor on the IR-GH release was undetectable. These results suggest that stimulation of the release and synthesis of GH mediated by the hypothalamic GRF fraction is under influence of the pool size of incubation media.

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Dihydrotestosterone-binding protein was observed in cytosols of the normal and hypertrophic human prostates. Association constant for dihydrotestosterone of cytosol of the normal prostate differed slightly from that of the hypertrophic prostate. However, maximum binding capacity was almost identical in these tissues. No distinct correlation was detected between proportion of glandular constituent and binding characteristics (Ka and maximum binding capacity) in the hypertrophic prostates.
Influence of some steroids on the binding with dihydrotesterone was examined. Testosterone, estradiol-17β, estriol, cyproterone acetate and SK & F 7690 inhibited the binding competitively. However, Sch 13521 did not exhibit any influence on the binding.

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Calcium excretion from rat intestine was measured by placing distilled water in an intestinal loop in situ and measuring the calcium content after various intervals. More calcium was excreted from the intestine of 18-month-old rats than that of 1-month-old rat. Acute hypocalcemia failed to change the intestinal calcium excretion significantly. Parathyroidectomy decreased intestinal calcium excretion and administration of Parathyroid Extract reversed it. Renal damage produced by injection of Na-sulfacetylthiazole increased the intestinal calcium excreation but dihydrotachysterol reversed it. Gastrin at 200μg/kg increased the intestinal calcium excretion. Calcium excretion, like calcium absorption, appears to be controled by various endocrine factors and the method of intestinal loop in situ appears to be useful to study the part played by these factors.

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Chronic subcutaneous injections with synthetic TRH (10μg/0.2ml of gelatine-saline) for 21 days slightly raised not only serum TSH but also serum LH concentration; those for 7 days tended to diminish the serum LH and FSH concentration. The magnitude of rise in the serum TSH was approximately equivalent to 1/5 of the value 30 days after a single injection with the same dose of TRH. The injections of synthetic LRH (5μg/0.2ml) for 21 days, on the other hand, raised the serum LH concentration 3.5 times as high as a single injection did. Pituitary TSH concentration was lowered conspicuously after TRH injections for 21 days; pituitary LH concentration was diminished remarkably after LRH injections for 21 days. Serum FSH concentration was not changed by the same LRH injection, while pituitary FSH concentration was diminished. Injections of TRH and LRH in combination for 7 or 21 days lowered the pituitary LH and FSH content more extensively than the separate LRH injection for 7 or 21 days. Some of our assay data may suggest the possible: synergistic or countervailing influences of the releasing hormones upon the secretion of the other trophic hormones. This is contradictory to the concept “one releasing hormone is responsible for one target cell”. This discrepancy may be explained by our working hypothesis as to the secretory cycle of the basophil.
It was tentatively concluded from our morphological manifestation that each releasing hormone did not act limitedly upon one target cell but universally upon a series of basophils. Both TRH and LRH exerted the analogous morphokinetic functions to convert the II-type cell (classical thyrotroph) into the III-type cells (classical LH-gonadotrophs) which are destined to turn into the III/IV-and IV-type cells (classical FSH-gonadotrophs), and to revert them to the original TI-type cells in the course of secretory cycle. There was a morphological evidence to show that the releasing. hormone might accelerate the rotation of secretory cycle of the basophil. Our working. hypothesis is that the various types of basophils may not be independent of each other, but modify their shape and property according to the different phases of secretory cycle, i. e., synthesizing, storing, secreting and resting phases. The II/III-and III-type cells were particularly accumulated after a long-term TRH injection; the IV/II-or II-type cells were accumulated but the IV-type cells were diminished in number after a separate LRH injection or the TRH and LRH injections in combination. The accumulation of the resting cells (IV/II-type) may account for the temporal stabilization of secretory cycle by the chronic injection.

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Serum 5α-dihydrotestosterone (DHT) and testosterone (T) were measured in 78 normal men aged between 12 and 89 years, separating DHT and T by celite microcolumn and using reliable radioimmunoassay.
In four age groups of young (20-39 years), middle aged (40-59 years) and old (60-79 years and 80-89 years) men, the mean±SEM for serum DHT were 98±12, 91±9, 82±7 and 54±15 (ng/100 ml), respectively. The corresponding values for T were 696±27, 698±18, 624±20 and 457±21 (ng/100ml). DHT and T showed significant correlation in each age group: r=0.625, p<0.05 (young men), r=0.727, p<0.02 (middle aged men), r=0.673, p<0.05 and r=0.734, p<0.02 (old men), respectively. There was a significant decline in DHT and T levels of 80-to 89-year-age group compared those of 30-to 39-year-age group (p<0.05 and p<0.005 respectively). But there was no significant changes in DHT and T levels between other age groups. After 3 daily im injections of 4, 000 IU human chorionic gonadotropin (hCG) the levels of serum DHT of the young group (20-32 years) and old group (72-78 years) increased 2.6 times and 1.9 times more, respectively. T increased 2.7 times and 1.4 times more, respectively.

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Female adult rabbits with coaxial bipolar electrodes chronically implanted in the brain were used. Evoked potentials (EVPs) were recorded in the medial basal hypothalamus (MBH) including the median eminence and the arcuate nucleus in response to stimulation of the dorsal hippocampus, medial amygdala and preoptic area. In the present experiment, the influence of ovulation-blocking drugs (pentobarbital, atropine and chlorpromazine) upon those EVPs was investigated.
Since short-latency responses (less than 4.5 msec), which were not consistently observed in those EVPs, were resistent to pentobarbital, they may be considered to be involved in the direct or pauci-neuronal pathways. These pathways were also not depressed in the excitability by atropine and chlorpromazine.
The efferent multi-neuronal pathways from the hippocampus to the MBH were influenced in different ways by pentobarbital, atropine and chlorpromazine, but the efferent multi-neuronal pathways from the amygdala to the MBH were depressed in the excitability by them. On the other hand, the EVPs elicited by preoptic stimulation was hardly affected by those drugs. These findings may suggest that the excitability of the neuronal pathways from the preoptic area to the MBH may be maintained against those agents.
In other words, pentobarbital, atropine and chlorpromazine differ in the manner of action upon the neural mechanism, in spite of their inhibitory effect upon ovulation.
The peak-latency of EVPs was scarcely influenced by those ovulation-blocking agents.
Finally, it is discussed that the multi-neuronal pathways could be concerned with the neural mechanism of copulatory ovulation and that the direct or pauci-neuronal pathways concerned with the electrically stimulated ovulation.

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Adult female New Zealand white rabbits with chronically implanted electrodes were used. Potentials (EVPs) were consistently evoked in the medial basal hypothalamus (MBH) including the median eminence and the arcuate nucleus by stimulation of the dorsal. hippocampus (HPC), medial amygdala (AMYG) and preoptic area (POA). These EVPs were variously influenced under endogenous or exogenous hormonal condition such as during estrus, after copulation and after hCG administration.
The MBH-EVP by HPC stimulation was depressed during estrus and was much more depressed after copulation than during estrus except the phasic facilitation at ten hours after copulation. The MBH-EVP by AMYG stimulation was significantly facilitated after copulation, nevertheless it was not so affected during estrus. The MBH-EVP by POA stimulation showed a reciprocal changes between during estrus and after copulation, that is, the facilitation in the former and the depression in the latter.
Namely, the excitability of the HPC-MBH neuronal system may be inhibited, that of the AMYG-MBH neuronal system may be facilitated and that of the POAMBH neuronal system may be facilitated and successively inhibited from the beginning of estrus to the accomplishment of the copulatory ovulation.
Finally, it was discussed that hCG directly might affect and depress the EVP representative of the neuronal pathways mentioned above.

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The chronological differentiation of the urovaginal septum of the female rat fetus and its critical period for the virilizing activity of testosterone were examined by measuring the urovagival septum length directly under microscope equipped with a micrometer.
Until the 18th day of gestation, definite differentiation of the urovaginal septum was not observed, but thereafter the septum developed rapidly and on the 21st day it was built up almost completely to separate the urethra and vagina.
Following a consecutive maternal subcutaneous administration of various doses of testosterone in olive oil for 4 days between the 17th and 20th day of gestation, a significant abridgment in urovaginal septum length was noted at a dose level of 0.01 mg/rat.
In the chronological examination, a significant inhibition of urovaginal septum development was observed 3 days after a single dose of 5 mg/rat of testosterone on the 17th day of gestation. When the same dose of testosterone was given on the 16th day of gestation, no effect was observed in the urovaginal septum length of female fetus on the 21st day of gestation. In the 17th-day-treatment group, however, a highly significant abridgment in the urovaginal septum was noticed compared with the control. Further abridgment was observed in the 18th day treatment group, and the 19th-day-treatment group showed the largest abridgment, but the length change in the 20th-day-treatment group was comparable to that in the 17th-day-treatment group. On these findings, it was concluded that the critical day of urovaginal septum abridgment by the virilizing activity of exogenous testosterone appeared to be the 19th day of gestation in female rat fetuses.

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The immunological and biological potencies of a new synthetic TRF analogue, L-pyroglutamyl-L-histidyl-L-pipecolic acid amide, were compared with those of TRF.In a radioimmunoassay system for TRF, parallel inhibition curves were obtained with TRF and the analogue, the immunological potency of the latter being approximately 50 per cent of the former.In vitro and in vivo TSH-releasing activities of the analogue were almost equal to those of TRF.As was observed with TRF, the in vitro TSH-releasing effect of the analogue was reduced in the presence of T₄, and the analogue was inactivated by incubation with rat serum.
The data suggest that the pyrrolidine ring of TRF can be replaced with piperidine ring without significant loss of the biological activity.

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Virilizing activities of nineteen steroids were examined by a method measuring the abridgment of urovaginal septum length of female rat fetuses directly under microscope, following oral administration of the steroids to the mothers for 4 days during the late period of gestation.
The characteristics of the virilizing activities of the steroids seemed to depend on their chemical structures.
No virilizing activities were observed in progesterone, retroprogesterone, chlormadinone acetate and nandrolone phenpropionate.The introduction of 6-methyl and 17-acetoxy groups into progesterone exhibited a marked virilizing activity.
With testosterone and its derivatives, the introduction of 17α-ethynyl group into testosterone slightly decreased the virilizing activity compared with the parentcompound.On the contrary, the introduction of [3, 2-C] pyrazole ring in addition to the saturation of A-ring strikingly increased the virilizing activity.The most active virilizing steroid in this group was stanozolol followed by oxymetholone, methyltestosterone and dimethysterone.Testosterone propionate was assumed to be less active than methyltestosterone by this route.
With 19-nortestosterone derivatives, the virilizing activities of compounds with 17α-ethynyl group were moderate, but the presence of 17α-ethyl group caused a marked increase of virilizing activity.While, nandrolone phenpropionate with long side chain at C-17 exhibited no significant virilizing activity.
The present results show the possibility of the evaluation on a potentialvirilizing activity of compounds in the female fetus, and also suggest that a dissociation between the virilizing activity and the androgenic activity may exist among some compounds.

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Levels of serum progesterone (P) and LH were determined daily in pregnant rats from Day 4 through parturition.In other pregnant rats the effect of antisera to LH (LH-A/S) and to LRF (LRF-A/S) on serum P, LH and fetal development were determined. These animals were treated with antisera on Days 8 through 10.Samples of blood were obtained on Day 8 before treatment started and again on Days 11 and 15.The animals were sacrificed on Day 15 and examined for fetal development and the ovaries weighed.
In untreated pregnent rats, the level of serum P showed two peaks, one on Day 7 and the other on Day 16.Serum P dropped acutely shortly before delivery.Serum LH showed no significant peaks through pregnancy but fluctuated between 30 and 160ng/ml.
In rats receiving 0.2ml LH-A/S s.c.on Days 8-10, the serum P was reduced to nondetectable levels on Day 11 and had recovered only slightly by Day 15. Pregnancy was terminated in all instances and the ovarian weights were reduced. Lower doses of LH-A/S were ineffective. Rats receiving 1.3ml of LRF-A/S on Days 8-10 showed no effect on serum P, or LH and no injurious action on fetal development.Lesser amounts of this LRF-A/S (0.8ml) were known to block the proestrous gonadotropin surge and inhibit ovulation in cycling rats.These findings confirm that LH is an essential component of the luteotropic complex during early pregnancy in the rat.They also suggest that a basal of LH and P can be to some extent maintained in rats treated with LRF-A/S during early gestation.

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Adult female rats show continual vaginal cornification and cease ovulation a few weeks after they are exposed to continuous lighting (light-estrous rats).When these rats were placed in the darkness for 10 hr, 80% of the animal ovulated approximately 46 hr later.Peripheral LH increased to a small peak immediately after placing in darkness concomitant with a decrease in pituitary LH content; a large peak, 20 times higher than the basal LH level, was observed at 20 to 22 hr.Progesterone concentration in ovarian vein blood remained at extremely low levels while estrogen levels tended to rise after small LH peak.This estrogen rise appeared to play an important role in inducing the main LH peak.Simulation of the small LH peak by low doses of exogenous LH succeeded in inducing ovulation in light-estrous rats in similar fashion to the exposure of light-estrous rats to 10-hr darkness.Therefore, the small amount of LH secretion observed after the initiation of the darkness-treatment may be considered as a trigger for the whole sequence of hormonal changes leading to ovulation.

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The effects of prostaglandin E₁ (PGE₁) and indomethacin (IDM) on the release of several pituitary hormones from the rat pituitary were investigated in vitro.
An addition of 2μg/ml of PGE₁ to the medium elicited the release of growth hormone (GH) and prolactin, but not of adrenocorticotropin (ACTH) and luteinizing hormone (LH).Although the addition of 1μg/ml of IDM alone resulted in no effect on the basal release of these hormones, IDM diminished the release of ACTH induced by crude rat hypothalamic extracts (HE) or lysine-8-vasopressin (LVP), and LH induced by HE or luteinizing hormone-releasing hormone (LH-RH).
These findings implicate that a part of PGE₁ action might be a direct one on the pituitary gland and PGE₁ might release GH and prolactin, whereas IDM might have a direct action on the pituitary gland, and that blunt the release of these pituitary hormones induced by several stimuli.

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Maternal blood and umbilical cord venous blood were collected immediately after delivery in 32 subjects who had completed normal birth during the 38 to 41 weeks of pregnancy, and the concentrations of human chorionic gonadotropin (hCG), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined by radioimmunoassay (RIA). The specific LH RIA utilized for the measurement of LH is unaffected by hCG at sample levels as high as 500IU/ml.
HCG concentrations were 10, 370±1, 480 mIU/ml (mean±SE) in mother's serum and 34.5±5.2ml in cord serum, while LH concentrations were 0.729±0.055 and 0.438±0.033ng/ml, respectively. The concentration of all 3 gonadotropins in maternal serum was greater than cord serum and the ratio was markedly high with 302: 1 for hCG, while the ratio was smaller for LH, being 1.7: 1, while that for FSH was 4.2: 1.There was no difference in the levels of LH and FSH in relation to the sex of the fetus.

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To investigate the initial response of the pituitary and adrenal cortex functionings of rats acutely exposed to cold, we developed a technique for cannulation which allowed sequential blood collection from conscious and freed rats.Two cannulae were inserted, one into the carotid artery used for collection and the other into the femoral vein for blood transfusion to keep the blood volume constant.Significant increases in plasma corticosterone and thyrotropin were evoked within 15 min after exposure.While plasma growth hormone (GH) slightly decreased, and the prolactin level tended to fall by 1 hr after cold exposure but the difference was not significant as compared with control values.

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Sera and fluid in molar vesicles from 4 patients with hydatidiform mole were analyzed for hCG and its subunits. These samples were initially chromatographed through a standardized Sephadex G-100 upward column.Starting materials and each fraction on gel filtration were radioimmunoassayed in homologous hCG, hCG-α and hCG-β assays.The specimens obtained from 4 patients contained primarily hCG and little hCG-β.Immunoreactive hCG-α was barely detectable in sera but was clearly demonstrated in vesicle fluid from molar patients.This immunoreactive hCG-α was combined with [¹²⁵I] hCG-β.The combined protein, [¹²⁵I] hCG and [¹²⁵I] hCG-β recombined with urinary hCG-α were concentrated in the superovulated rat ovary in vivo.However, [¹²⁵I] hCG-α and [¹²⁵I] hCG-β were not taken up by the ovaries.The biosynthesis of hCG and the possible biological role of hCG-α are discussed.

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