New test systems for evaluating LH and LRF activities using a combined local techniques of rat testis were presented.
A response brought by intratesticularly injected LH was assessed by testosterone output in the testicular vein blood (TVB). A significant rise in testosterone output was obtained with 5 ng of NIH-LH-S15 and 1/5, 000 gland equivalent dose of rat pituitary extract. As a sensitive bioassay for LH, this test system seems promising.
For investigating LRF activity, a rat stalk-median eminence extract (rSME) was locally infused into the testis bearing rat or quail pituitary implant. LH contents in pituitaries of pre-and post-implantation (2 hr) were measured by intrabursal OAAD test for evaluating a direct effect of LRF, and at the same time the indirect effect due to the released LH from the implanted pituitary, was assessed by the increase of testosterone in TVB. Effects of intratesticular implantation of pituitary alone on these two parameters were also investigated.
It was noteworthy that the internal medium of rat testis induced a striking LH release from the rat pituitary, but not from the quail pituitary. A factor (s) other than ionic components in the testis (tentatively named as ITLR) may be responsible for this release.
Upon the quail pituitary, rSME mainly manifested the LH releasing activity. Although the LH releasing activity of rSME on the rat pituitary was overshadowed due to concurrent action of ITLR in this intratesticular test system, the stimulatory effect of rSME on the LH production was clearly indicated.
Species specificity and action mode of LRF were discussed from these findings.

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Mammary gland development and secretion in male rats were induced by single injections of reserpine. The lactogenic effect of reserpine was most marked when a dose of 1mg/kg was given. The alveoli and ducts of the male mammary glands were greatly distended with milky secretion. Similar changes in the glands were also observed but in a smaller number of animals when a dose of 0.5-0.75mg/kg was given. Nialamide (250mg/kg) counteracted the effect of reserpine (1mg/kg) when given 16-18 hours before the reserpine injection. A similar inhibition was obtained by L-DOPA (250mg/kg) when injected one hour after the reserpine injection. These results provide the evidence that the male pituitary is capable of secreting prolactin in response to reserpine, and also suggest the involvement of monoaminergic mechanisms in the process of prolactin release in the male rat.

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The changes of growth hormone-containing cells during the tumorigenesis and subpassages of the estrogen-induced pituitary tumors were studied with peroxidaselabeled antibody method.
In a primary pituitary tumor induced with estrogen in a female W/Fu rat, growth hormone-containing cells were rather small and compressed away by the tumorous growths to localize mainly to the peripheral areas, corresponding with the unchanged acidophils in the routinely stained sections.
The cells positively stained with the peroxidase-labeled antibody were scattered in a transplantable mammotropic pituitary tumor (MtT/F4). Since this tumor is well known to produce growth hormone in addition to prolactin and adrenocorticotropic hormone, a few non-functional growth hormone-containing cells which remained in the primary pituitary tumor were considered to have transformed to secrete growth hormone during the further subpassages.
Further, the changes and variations of the hormonal activities of the estrogeninduced pituitary tumors were discussed from the view point of the different cellular populations.

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Incubations of rat ventral prostate with testosterone-14C and 5a-dihydrotestosterone (5α-androstane-17β-ol-3-one)-³H revealed the gradual increase in uptake of both steroids into the tissue. Ratios of ³H/¹⁴C of the incorporated steroids were similar during the incubation periods, and this indicated that incorporation of both the steroids proceeded in the same pattern. Radioactivity in purified 5α-dihydrotestosterone from the incubated whole tissues showed a gradual increase in both ¹⁴C and ³H, and ³H/'⁴C ratio was also almost constant throughout the incubation period. Therefore, rate of the conversion of testosterone to 5α-dihydrotestosterone seems to be constant during the incubation.
A considerable difference in ³H/¹⁴C ratios of ethyl acetate extracts was observed between the various subcellular components after 30 min incubation. ³H/¹⁴C ratio in 5α-dihydrotestosterone, however, showed a small difference in all subcellular components. Most of the radioactivity incorporated into purified nuclei was attached to 5adihydrotestosterone.

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A light and electron microscopic observations on pancreatic islets of spontaneous diabetic mice (yellow KK) were performed. Degranulation and glycogen deposition of B cell observed at the age of 5 weeks were followed by hypertrophy and central cavitation of islets. These changes became prominent until they were 16 weeks of age, and then degranulation and glycogen deposition became diminished with increasing age. By the electron microscopy, degranulated B cells were found to contain well developed Golgi apparatus, abundant granular endoplasmic reticulum and fine granules of glycogen. B granules were occasionally fused to cell membrane to represent emiocytosis. These changes show increased activities of synthesis and release of insulin which corresponded with high levels of plasma immunoreactive insulin. During the phase of hypertrophy of islets, there were observed acinar typed cells containing B granules in islets, and aldehyde fuchsin positive cells in pancreatic ducts. These phenomena suggest the validity of the transformation of extra-insular cells into B cells in the process of hypertrophy of islets.

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KK mice fed on semisynthetic diets with a usual composition developed marked obesity and diabetic symptoms in comparison with those kept on laboratory chow. The observed changes included obesity, fatty liver, glucosuria, hyperglycemia and degranulation of pancreatic B cells. These changes became more prominent with prolonged period of feedig up to 24 weeks. Among them, fatty liver and obesity were also induced in control ICR mice upon feeding semisynthetic diets but the developments were limited in moderate degree and no diabetic symptom developed. These results indicate that the different response to diets in both mice can be attributed to genetic factors.

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A correlation between the morphology of mature follicles and estrogen secreting activity of ovaries was investigated on the proestrous rat before and after the exposure of endogenous ovulating hormone or ovulatory dose of exogenous gonadotropins. Histology of mature follicles until 5 p.m. in proestrus, when estrogen secretion was highly stimulated, was characterized by a granulosa layer with smooth inner surface, in which many mitoses were seen, regularly arranged nuclei of basal granulosa cells, and relatively thin theca interna sharply bordered from granulosa layer. During the process of ovaries losing estrogen secreting activity after exposure of ovulating hormone, a slight thecal swelling with hyperemia, accumulation of intercellular fluid in granulosa layer were recognizable in mature follicles. At 11 p.m., when estrogen secretion had fallen to the lowest level, orientation of nuclei in basal granulosa cells became irregular and a sharp border between granulosa layer and theca layer became indistinct. It was also noticed that the glucose-6-phosphate dehydrogenase activity distributed in theca interna decreased slightly at this time. If FSH or LH was administered 6 hr before the normal time of ovulatory surge, the same histological and histochemical changes described above were advanced approximately 6 hr. In addition, the minimal doses of gonadotropins required for causing such morphological changes in mature follicles were exactly the same as those to induce termination of estrogen secretion and ovulation. These findings support our previous explanation for the mechanism by which gonadotropin causes termination of estrogen secretion in proestrus, i.e. ovulation inducing hormone initiates the differentiation of follicular elements towards ovulation, and during the process of differentiation, unfavorable conditions may be brought about for estrogen synthesis.

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Progesterone and 20α-hydroxypregn-4-en-3-one (20α-OH-P) secretory rates of rat ovaries were measured during pregnancy and pseudopregnancy. In the pregnant rats, 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) activity of the ovaries, wet weights of corpora lutea and their concentrations of cholesterol, progesterone and 20α-OH-P were also determined. Progesterone secretion increased but fluctuated during pregnancy: a small rise on Day 4 was followed by a fall on Day 7, and a marked rise on Days 13 to 15 was followed by a great fall on Days 20 to 23 (the day sperm was found in vaginal smears was designated as Day 1). 20α-OH-P secretion decreased throughout pregnancy, although a small rise appeared around Day 15. The second peak of progesterone secretion and the small peak of 20α-OH-P occurred in coincidence with the growth of corpora lutea which had remained fairly constant by Day 10. After the cessation of corpora lutea growth on Day 16, both progestin secretions turned to decrease. Cholesterol concentration in corpora lutea, which remained relatively low during the first half of pregnancy but gradually increased with a gentle slope by Day 17, suddenly doubled the value on Day 18 when progesterone secretion kept decreasing. Progesterone secretion continued to decrease until the day of parturition (Days 22 to 23). 20α-OH-P began to increase on Day 22 in accordance with the increase of 20α-OH-SDH activity in corpora lutea, which had never appeared before Day 20. After parturition, progesterone secretion began to increase on the third day, conversely with a rapid drop in 20α-OH-P secretion in lactating rats. Corpora lutea progesterone, which showed the highest value on Days 5 to 8 of pregnancy followed by a gradual decrease to the lowest on Day 23, did not appear to play a special part in the regulation of progesterone secretion. In pseudopregnancy, progesterone secretion increased beginning 3 to the maximum on Day 7 and declined thereafter to Day 14, while 20α-OH-P secretion decreased during the same period of time, but the secretory rate was generally higher than that in pregnancy, so that a higher ratio of progesterone to total progestin (progesterone plus 20α-OH-P) was obtained in pregnancy than in pseudopregnancy.

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An increase in the ovarian secretion of progesterone occurring on the afternoon of the day following ovulation was observed even in the rats hypophysectomized after ovulation, confirming the automaticity of newly formed corpora lutea secreting progesterone independently of pituitary function. Either estrogen (estradiol, estradiol benzoate) or prolactin (NIH-P-S8) given shortly after ovulation prolonged the rise of ovarian progesterone secretion as well as the duration of diestrous phase in vaginal smears. When rats were hypophysectomized preceding the hormone treatment, estrogen was not effective but prolactin was effective to cause luteotrophic action (prolongation of increased progesterone secretion), indicating that estrogen acts indirectly on the ovaries by stimulating the secretion of luteotrophic substance from the pituitary. 20α-Hydroxypregn-4-en-3-one secretion, which is known to be almost parallel with progesterone secretion in the normal diestrous stage, was lowered during the period when exogenous estrogen produced luteotrophic action to sustain the increase of progesterone secretion.

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An injection of ACTH caused a remarkable stimulation in the rate of. ³H-choline incorporation into ethanol-ether extractable fraction of the subcellular components from rat adrenal glands as early as 3.5hr after the injection. The rate of increase in the incorporation due to ACTH administration was most conspicuous in the inner membrane fraction of mitochondria; in this fraction almost 500% increase was observed. Administration of cycloheximide in amount that blocks protein synthesis prior to ACTH treatment inhibited the ACTH effect on the choline incorporation completely, while a large dose of Actinomycin D did not show any influence on the ACTH action.

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Thyroglobulins were extracted from the thyroid glands of one amphibia (Xenopus laevis), one ayes (chicken) and five mammals (whale, rat, hog, monkey and human), and purified by means of salting-out with ammonium sulfate followed by DEAE-cellulose chromatography. Sedimentation coefficients, electrophoretic mobilities, isoelectric points, immunochemical properties as well as iodine contents and amino acid compositions of all or most of these thyroglobulins were determined and the results were compared with each other. In spite of a great variety of animal species studied, these thyroglobulins were found to be rather similar in various respects, while species specificity was clearly indicated. Most distinct differences were observed among amphibian, avian and mammalian thyroglobulins, especially in amino acid composition, isoelectric point and immunochemical properties.

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Inheritance of insulin sensitivity has been studied on C57BL, KK and their F₁-hybrids with or without the yellow obese gene (A^y).
Intraperitoneal injection of insulin was followed by a remarkable fall of the blood glucose in C57BL, but a slight fall in KK. In vitro, the adipose tissue of C57BL showed more remarkable response to insulin in glucose-1-¹⁴C oxidation than that of KK. F₁-hybrid clearly showed intermediate sensitivity to the hormone between the parental strains both in vivo and in vitro.
The yellow hybrid, A^y (the yellow obese gene)-carrying F₁-hybrid, showed adiposity and hyperinsulinemia, but did not develop the diabetic symptoms observed in yellow KK, A^y-carrying KK, such as hyperglycemia and degranulation of pancreatic B cells. In the hormone sensitivity of adipose tissue, yellow hybrid was less sensitive than nonyellow hybrid, but more sensitive than KK or yellow KK.
These data suggest that the insulin resistance of KK may be determined by a dosage of polygenes, and may result in higher susceptibility to diabetogenic action of the obesity caused by A^y gene.

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Synthetic thyrotropin-releasing factor (TRF), L-pyroglutamyl-L-histidil-L-prolineamide, has been administered intravenously to 35 cases of normal or suspected pituitary insufficient volunteers in doses ranging from 25 to 200μg. Changes of plasma TSH levels, PBI and T₃ resin sponge uptake (T₃RSU) were estimated. TRF in doses of 25-200 μg stimulates a rapid rise in plasma TSH in all of the 13 normal subjects. 3 out of 5 normal subjects showed rise in serum PBI while T₃RSU remained unchanged. The only side effect was transient nausea in several cases. No or slight rise in plasma TSH levels occurred followine 100μg of TRF in patients with pituitary chromophobe adenoma and craniopharyngioma who had a longstanding pituitary insufficiency and low level of thyroid function. However, certain cases of both tumor showed almost normal TSH response to TRF. It was also noticed that no TSH response to TRF was observed in two hyperthyroid patients and one medicated with thyroid powder. Normal TSH increase following TRF was obtained from cases with suspected hypotituitarism, diabetes insipidus, arachnoiditis of chiasma region, myotonic dystrophy and a 77 years-old female. Suboptimal response were observed in a patient with suspected Cushing' syndrome. It is concluded that synthetic TRF is useful for testing the pituitary TSH reserve in man. Plasma TSH response to TRF in various clinical conditions was discussed.

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Medio-basal hypothalamic lesions were made by electric coagulation in Sprague-Dawley strain rats 20-24 hours after birth. Testicular development in these animals was examined 20, 40 and 60-70 days after birth. Transection (Group 1) or severe lesion (Group 2) of the pituitary stalk caused arrest of both spermatogenesis and testicular development. In these animals, on the 40th and 60th day after birth, the seminiferous tubules were very small in diameter and no spermatids or spermatozoa were found in the lumen. The seminiferous epithelium was very thin, and spermatogenesis was arrested in the premeiotic stage. Lesion of the posterior part of the median eminence, including the stalk (Group 4), also induced testicular atrophy, but spermatogenesis was not disturbed. Damage of the anterior portion of the median eminence (Group 3) did not cause testicular atrophy. When the median eminence and the stalk were intact, lesions of the hypothalamus, including the subthalamus, mamillary body and half the hypothalamus (Group 5), did not depress spermatogenesis. The hypophysis did not show any degeneration in all cases examined, although the size was very small in Groups 1 and 2. These findings suggest that development of testiculotropic function of the hypophysis is dependent on the hypothalamus, especially on the posterior medio-basal hypothalamus, even though the hypophysis grows independently of hypothalamic control.

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A theory to understand the total performance of complicated reproductive functions in mammals and to explain its integrative mechanism is presented with special reference to its mathematical formulation and digital computer simulation. Functional connections of components and subcomponents in the brain-hypophyseal-gonadal system are described through evaluation of experimentally established facts and our own hypotheses. Input-output relations of informations carried by neural spikes, 3 kinds of neurohormones and 3-5 kinds of hypophyseal and gonadal hormones are expressed in graphical, literal and mathematical terms. Sexual and species differences in both function and structure are also dealt with. The brain of both sexes is assumed to contain 3 different functional subcomponents such as neural, neurosecretory and store mechanisms. The neural mechanism is composed of detectors for photoperiodicity, copulatory stimulation and blood levels of hormones, and neurons which send either stimulatory or inhibitory signals to regulate the production and release of neurohormones. It is also assumed that each hormone can exert a positive feedback effect on the brain at first for a short period activating the stimulatory neural mechanism to be concerned, and then a negative effect for a longer period through the inhibitory mechanism. If the threshold, time constant and output level of each detector and neuron are adequately determined in response to neural or hormonal inputs, it is suggested that the secretory pattern of gonadotropins and sex hormones may be cyclic as in most female species and superficially non-cyclic as in the male. On the basis of such assumptions, a general principle for the regulatory mechanism is theoretically synthesized. This is exemplified by chronological changes in the dynamic state of the 4-day estrous cycle in the rat. This theory seems to be well evidenced by a result of numerical analyses by computer, showing a considerably good accordance with the performance of the real system.

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The Igarashi-McCann method, reported in 1964 as a new biological method assaying FSH with MED corresponded to 2μg equivalents of NIH-FSH-Sl utilizing intact female mice of Swiss-Albino, would be appreciated to be indispensable for sensitive evaluation of FSH, in spite of having been suffered from poor precision which had been destined with the principle of uterine weight augmentation response. The optimum dose of HCG to be added was confirmed to be 0.075 iu through variance analysis of regression line and statistical investigation of linearity and stability in the dose response curve. The limitation and unification of beginning body weight of recipient were concluded to be inevitable not only for improvement of precision but also for qualitative unification in uterine weight response. Utilizing the unified recipient of 8 g, the stable and specific estimation of FSH in normal urine and plasma can be performed within such levels of maximum permitted contaminating dose (MPCD) as 2.0μg equiv. of NTH-LH-S13, 2.5μg equiv. of NIH-GH-S7, 1.0μg equiv. of NIH-GH-HS1082B, 400 mu equiv. of NIH-P-S7, 200mμ equiv. of Armour-ACTH, 200 mu equiv. of NIH-TSH-S5, and 0.005 μg of Estradiol with 0.075 iu of HCG, after referring reported contents of these hormones in pituitary, in plasma, and in urine. In conclusion, the results of Igarashi-McCann assay are specific for FSH, if less than 14.4 μg of rat anterior pituitary extract or less than 2.0ml of rat or human plasma, or 1.0 hr and 0.2 hr equiv. or less of non-pregnant human urinary extract except of acromegaly is injected into one mouse, respectively.

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The hypophysial stalk of the horse had a typical structure of the median eminence and contained noradrenaline, dopamine, serotonin, vasopressin and paraldehyde fuchsine-stainable material. The posterior wall of the hypophysial stalk contained higher concentration of noradrenaline than the anterior and lateral walls. On the other hand, the anterior and lateral walls contained dopamine but the posterior wall did not. The concentration of serotonin was almost the same through these three portions. Vasopressin concentration was highest in the anterior wall and lowest in the posterior wall. Large droplets of paraldehyde fuchsine-stainable material were accumulated in restricted areas of the anterior and posterior walls. Fine droplets of the material were distributed in the outer layer of all the three portions of the stalk. These results indicate that there is the regional and functional differentiation in the median eminence of the horse.

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In order to obtain a more homogeneous preparation of human placental lactogen (HPL), a proteo-hormone which chiefly possesses lactogenic activity, the extraction and the purification of this protein from human placental tissues was carried out by a modification of the method of Friesen (1965a, b). The extracts of minced placenta were made to be acetone-dried powder. The powder was extracted with water and then the soluble portion was precipitated with half saturated ammonium sulfate. This crude preparation was fractionated on ECTEOLA-cellulose column and Sephadex G-100 equilibrated with 0.05M ammonium bicarbonate, pH8.3. The preparation thus obtained was electrophoretically homogeneous and showed prominent lactogenic effects in the pigeon crop sac assay. No gonadotropic activity of this protein was observed.

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